Recent advances and perspectives in molecular epidemiology of Taenia solium cysticercosis

Highlights

• Sympatric distribution of Taenia solium, Taenia saginata and Taenia asiatica in Asia.

• Inter-species outcrossing between T. saginata and T. asiatica.

• Two, Asian and Afro/American, genotypes of T. solium.

• Importance of comparative studies of mitochondrial (haploid) vs nuclear (diploid) genes.

• Intra-species outcrossing between two genotypes of T. solium in Madagascar.

Abstract

Cysticercosis caused by accidental ingestion of eggs of Taenia solium is spreading all over the world through globalization and is one of the most neglected, neglected tropical diseases (NTDs) or neglected zoonotic diseases (NZDs). In the present study, the reason why T. solium cysticercosis has been neglected is discussed at first, and followed with an overview on the most recent advances and perspectives in molecular approaches for epidemiology of T. solium taeniasis/cysticercosis, since although taeniasis does not constitute recognized zoonoses, transmission and complete development are dependent on human definitive hosts. Main topics are discussions on (1) the two, Asian and Afro/American, genotypes of T. solium, (2) comparative analysis of mitochondrial (haploid) and nuclear (diploid) genes, and (3) the presence of hybrids of these two genotypes which indicates out-crossing of two genotypes in hermaphrodite tapeworms in Madagascar. Additional topics are on (4) the usefulness of phylogeographic analyses to discuss where the infection was acquired from, and (5) miscellaneous unsolved topics around these genetic diversity of T. solium.

Introduction

Cysticercosis (CC) including the most potentially lethal neurocysticercosis (NCC), ocular cysticercosis (OCC), subcutaneous cysticercosis (SCC) etc. is caused by the presence of larval stage, cysticercus or cysticerci of the pork tapeworm, Taenia solium after accidental ingestion of eggs released from adult tapeworm carriers or autoinfection with eggs in the tapeworm carriers themselves (Fig. 1, Fig. 2). Disseminated cysticercosis cases are caused mainly in tapeworm carriers themselves by autoinfection even if detection of tapeworm(s) is not always easy (Schantz et al., 1983, Schantz et al., 1998, Margono et al., 2002, Kobayashi et al., 2013, Ito et al., 2014). The only definitive host of T. solium is human, Homo sapiens (de Queiroz and Alkire, 1998, Hoberg et al., 1999, Hoberg et al., 2001, Hoberg, 2002, Hoberg, 2006, Michelet and Dauga, 2012, Zarlenga et al., 2014a, Zarlenga et al., 2014b). Adult tapeworm matures in the human intestine after eating uncooked pork contaminated with cysticercus or cysticerci. The life span of T. solium tapeworm is difficult to observe, but is probably within a few years (Yoshino, 1934, Lightowlers, 2013) and critically differs from that of Taenia saginata for over 30 years (Pawlowski and Schultz, 1972, Pawlowski, 1982). The size and thickness of the gravid proglottid as well as the whole worm critically differ between T. saginata or Taenia asiatica and T. solium. T. solium is much smaller and thinner than T. saginata or T. asiatica (Fig. 3).

Although the eggs are infective not only to swine and humans but also dogs, and the range of potential intermediate hosts is broader than swine and dogs (Hoberg et al., 2001, Ito et al., 2002a), the life cycle of this parasite is usually completed between humans (tapeworm carriers, definitive host) and pigs (intermediate host). Therefore, T. solium is now considered to be the most important foodborne parasite globally (Robertson et al., 2013, Robertson et al., 2014) and so, rationally eradicable disease (Schantz et al., 1983, Pawlowski et al., 2005, Flisser et al., 2006, Pawlowski, 2006, Pawlowski, 2008, Schantz, 2006, García et al., 2007, WHO, 2010) but difficult due to poverty, lack of education, traditional culture, etc.

In cysticercosis endemic areas in Asia or perhaps in Africa and Americas, pigs and dogs are living together and free roaming. Although our caution towards the control of cysticercosis has been focused on humans and pigs, dogs should be included as risk factor for both humans and pigs which may cause additional life cycle between dogs and humans, and confusion in pigs contaminated not with T. solium but with Taenia hydatigena (Ito and Budke, 2014, Ito et al., 2014, Ito, 2015, Wandra et al., 2015).

Dogs are the definitive host of T. hydatigena and several other Taenia species (Table 1) (Nakao et al., 2013). Among these Taenia species, T. hydatigena is the commonest species in livestock. Therefore, pigs are commonly infected with cysticercus/cysticerci of T. hydatigena. It might be more common than cysticercus/cysticerci of T. solium in free roaming pigs where dogs and pigs are living together (Ito et al., 2014, Wandra et al., 2015). This has often been ignored, but many epidemiological survey of pigs where dogs are living together should remind this fact in order to obtain sound “direct evidence of infection”. Recent waves to use antigen-ELISA without necropsy of pigs are difficult to evaluate if antigen-ELISA positive pigs really include pigs infected with T. solium, since pigs infected with T. hydatigena become strongly positive (Fleury et al., 2003, Dorny et al., 2004). Without necropsy of pigs to confirm infection with T. solium, it is not clear whether the antigen-ELISA positive results are from contamination with T. hydatigena, since the monoclonal antibody was prepared against metacestodes of T. saginata and is highly possibly cross-reactive to other Taenia species (Brandt et al., 1992, Fleury et al., 2003, Dorny et al., 2004, Ito, 2013, Ito, 2015, Ito et al., 2014). Furthermore, local people in many Asian countries love to eat dog meat (Hoberg et al., 2001, Ito et al., 2002a, Ito et al., 2005, Ito et al., 2014, Ito and Budke, 2014). Therefore, there may be additional alternative but minor transmission of T. solium between dogs and humans (Ito et al., 2002a).

Pigs are scavengers and get infection with eggs of T. solium from human feces and those of T. hydatigena from dog feces, whereas dogs are scavengers and get infection with eggs of T. solium from human feces (Ito et al., 2002a). So far we know, there is no report stressing the importance of dogs in cysticercosis endemic areas which interferes the sound prevalence of T. solium in pigs. The importance of dogs which contributes the endemicity of taeniasis/cysticercosis is, therefore, stressed in order to grasp the risk factors in humans and pigs (Ito et al., 2014).

The risk factors in pigs crucially differ in pigs reared under different conditions. (1) Pigs reared in indoor in endemic areas or in door farming in developed countries are basically free of cysticercosis except the environment is accidentally contaminated with eggs of T. solium. Laborers harboring T. solium tapeworm(s) from endemic areas or countries are high risk of accidental contamination of pigs (Yanagida et al., 2012). Monitoring of antibody responses or circulating antigens is highly useful, since T. solium is only species which contaminates pigs. In contrast, (2) pigs have high risk of infection with eggs of T. solium and T. hydatigena from human and dog feces, respectively, where pigs are reared out door with dogs. In this situation, more specific serology should be introduced to differentiate pig contaminated with T. solium from T. hydatigena. The latter is very common in endemic, remote areas in developing countries all over the world (Ito et al., 2014).

In this review article, main topics are molecular approaches for detection and identification of the pathogens in human, pigs and dogs for epidemiological studies.

T. solium was considered to be distributed mostly in rural and remote areas coincidental with regions where people under economic poverty consumed pork without meat inspections (Fig. 2) (Robertson et al., 2013, Robertson et al., 2014, Ito and Budke, 2014, Ito et al., 2014). However, T. solium tapeworms have rarely been confirmed even through microscopic examination of feces for the soil transmitted helminthiases (STHs) (Carney et al., 1980, Saksirisampant et al., 2002, Belizario et al., 2004, Kepha et al., 2015, Greenland et al., 2015, Ngui et al., 2015). In most of STH surveys, there is no record of Taenia eggs. It is mainly because Taenia eggs are not constantly distributed in stool samples, but they are abundant only when gravid proglottids(s) were discharged in tapeworm carrier’s intestine. The mechanism of discharging of gravid proglottid(s) remains unsolved, although apoptosis may be involved in. Most recent study of STHs with molecular tools is interesting (Gordon et al., 2015). Although three human Taenia species have been reported in the Philippines (de Leon, 2005, Yamane et al., 2013), Gordon et al. (2015) detected T. saginata only. It might include both T. saginata and T. asiatica or all might be T. asiatica. Distribution of human Taenia in the Philippines may be highly variable among the islands, since people living on each island have their unique culture and different religion, etc. Further studies are necessary to get better understanding of the transmission of human Taenia in this country.

In the mass drug administration (MDA), the priority of mass treatment for deworming is directed against STHs, food-borne trematodiases (FBT) and schistosomiasis. Besides, it is impossible to morphologically identify the species for Taenia eggs under microscope. Therefore, almost all field works on helminthiases by microscopy tend to describe just Taenia eggs without any identification of the species. Molecular identification is essential for Taenia egg(s) (Nkouawa et al., 2009, Nkouawa et al., 2010) but is thus far mostly impossible in rural or remote areas where epidemiology of STH, FBT and schistosomiasis is carried out (Nkouawa et al., 2012). Furthermore, deworming of Taenia spp. in MDA has some problems. If praziquantel (PZQ) > 10 mg/kg B.W. is administered to tapeworm carriers and if the species is T. solium, PZQ may cause sudden seizure attack, acute symptomatic neurocysticercosis (NCC) within a half day, since higher dose of PZQ has metacestocidal effect and the damage of living cysticerci may result in acute allergic responses (Flisser et al., 1993, Sarti et al., 1994, Sarti et al., 2000, Willingham et al., 2008, Ito et al., 2013). Therefore, treatment with corticosteroid is essential to avoid accidental seizure attacks after high dose of PZQ treatment (García et al., 2004, García et al., 2014, García et al., 2015, Willingham et al., 2008, Del Brutto and García, 2013, Del Brutto, 2013, Del Brutto, 2014a, Del Brutto, 2014b, Nash et al., 2015). However, such complicated treatment is not suitable for MDA (Ito et al., 2013). Alternatively, PZQ < 10 mg/kg has been recommended for tapeworm carriers in T. solium endemic areas which usually does not damage larval stage of T. solium.

However, it is difficult or impossible to obtain such information where T. solium is endemic until unexpected NCC cases are reported. In contrast, among the three human Taenia species, T. saginata, the beef tapeworm, is more common and much easier to be detected by tapeworm carriers themselves, since a single gravid proglottid separated from whole worm actively escapes from anus to the outside any time of almost every day. In one volunteer’s case, he experienced more than 30 proglottids coming out from his anus within one day (Ito’s own observation).

T. saginata is well known to be common in local people in Bali, Indonesia (Suweta, 1991, Depary and Kosman, 1991 Theis et al., 1994, Simanjuntak et al., 1997, Margono et al., 2002, Margono et al., 2005, Wandra et al., 2006, Wandra et al., 2015). Throughout our field work in Bali from 2002 until 2010, all tapeworms discharged from more than 100 tapeworm carriers were definitively identified as T. saginata (Wandra et al., 2015). Wandra and his colleagues treated T. saginata taeniasis patients using PZQ, 15 mg/kg B.W., with no expectation of asymptomatic NCC. However, after discharge of T. saginata by PZQ treatment, unexpected sudden seizure attacks were observed in several tapeworm carriers. From such work, double infections with T. saginata adult tapeworm(s) in the intestine and T. solium cysticerci in the brain have been confirmed (Sudewi et al., 2008, Wandra et al., 2013, Wandra et al., 2015). Such situations may be common in almost all countries in Southeast Asia, and if low dose of PZQ would be administered for such tapeworm carriers, one could overlook the veiled asymptomatic NCC cases (Flisser et al., 1993, Sarti et al., 1994, Sarti et al., 2000).

In many areas where taeniasis has been reported, we are therefore facing a problem how to identify the species and how to treat taeniasis patients. In remote areas, low dose of PZQ is recommended for treatment of tapeworm carriers with no expected accidental epileptic attacks (Flisser et al., 1993, Sarti et al., 1994, Sarti et al., 2000, Wandra et al., 2013). However, it is impossible to obtain any information on the endemicity of hidden NCC without any accidental seizure attacks. In rural areas on Bali where access to the capital city is not difficult through semi-urbanization other than the endemic rural areas, high dose of PZQ may be recommended, because the on time treatment of seizure attacked patients is possible. Or alternatively, we recommend introduction of serology for cysticercosis in screening of taeniasis carriers and STHs in order to detect asymptomatic NCC based on the recognition that cryptic infections are endemic and potentially most lethal parasitic disease on Bali (Margono et al., 2002, Sudewi et al., 2008, Wandra et al., 2015). When some people are confirmed antibody positive to diagnostic antigens for cysticercosis (Ito et al., 1998, Ito et al., 2014, Sako et al., 2013, Ito and Budke, 2014), they are recommended to admit big city hospital(s) for further treatment. However, there is a financial problem how to cover the admission fees!

In conclusion, there is almost no data showing the real distribution of T. solium and epidemics of NCC without any accidental detection of symptomatic NCC cases in the world including developed countries (Margono et al., 2000, Margono et al., 2005, Yamasaki et al., 2005, Yanagida et al., 2012, Kobayashi et al., 2013).

Even if there are no such NCC cases and taeniasis cases are due to T. saginata or T. asiatica, we have to note that asymptomatic NCC could remain endemic, cryptic and neglected. This is the real present situation of cysticercosis in humans in Asia at least (Ito et al., 2014, Wandra et al., 2015) and may be the same in other areas. Endemicity of NCC or distribution of T. solium is exclusively and accidentally recognized by detection of cysticercosis, mainly symptomatic NCC cases. Furthermore, through globalization and advances in diagnostics, symptomatic cysticercosis in humans may become more common not only in developing countries where people eat pork but also in developed countries where people do or do not eat pork (Fig. 2) (Schantz et al., 1998, Al Shahrani et al., 2003, Hira et al., 2004, Sorvillo et al., 2007, Sorvillo et al., 2011, Leshem et al., 2011, O’Neal et al., 2012, Fabiani and Bruschi, 2013, Robertson et al., 2014, Ito and Budke, 2014, Ito et al., 2014, Ito, 2015, Wandra et al., 2015, O’Keefe et al., 2015). People in capital cities in high-income countries are not free of risk of NCC any more (Fig. 2) (Schantz et al., 1992, Hira et al., 2004, Leshem et al., 2011, Khan et al., 2011, Yanagida et al., 2012, Del Brutto, 2013). An increasing risk factor for transmission and infection relates to globalization of an immigrant work-force originating in countries where T. solium is endemic.

It is well known that cysticercosis is focal and local disease where people kill pigs in the backyard of owner’s home, and pork with or without contamination of cysticerci is only meat for the family members, relatives and neighbours for special ceremonies. Under very poor conditions, they cannot stop eating pork, since it is only special meat available for the family members with no money to buy pork (Ikejima et al., 2005, Li et al., 2006). Pigs, either contaminated with T. solium or not, reared by the poor families are the only meat source for the family members.

Much wider situation of populations at risk is by consuming pork through rough meat inspection in endemic areas where economy of the populations is a little bit or much better than the poorest populations. If owners were heavily penalized when their pigs were identified to be contaminated with T. solium, they used to sell their pigs under dark or bush without meat inspection, and recommend preparation of “delicious” sausage, etc. Or they asked the officials not to declare contaminated pigs under the table. Corruption is another human attitude, and may be common not only in Asia but also in other areas. More important is that it is difficult to check whole meat at all (Suweta, 1991) (Fig. 4). Pork with a very small number of cysticerci of T. solium may easily be judged “safe” without additional serological tests.

Travelers who are interested in visiting remote areas, and people visiting family members and relatives in the remote areas in their home countries in Latin America and Southeast Asia, etc. may get infection with tapeworms of T. solium as well after eating local dish (Yanagida et al., 2010, Jongwutises et al., 2011). These are populations with risk of taeniasis. Then, such taeniasis carriers themselves become autoinfected with eggs (Schantz et al., 1983, Schantz et al., 1998, Margono et al., 2002, Kobayashi et al., 2013) and simultaneously contaminate others who have intimate contact with the carriers themselves etc. (Schantz et al., 1992, Allan et al., 1996, Ikejima et al., 2005). Thus far, cysticercosis as cryptic NTD is common in developing countries where pork is consumed. Cysticercosis in general including different types of cysticercosis may be more cryptic. Therefore, we better stress NCC but not cysticercosis as higher impact from public health. Pawlowski pointed out that “Control of T. solium infection is justified exclusively by severe pathology caused by neurocysticercosis in humans. Therefore, from medical point of view, it would be more appropriate to use the term control of taeniasis/neurocysticercosis (T/NCC) rather than the traditional one control of taeniasis/cysticercosis (T/CC). The formal change of the term would strengthen the clinical justification of the control and also turn an attention to the fecal-oral way of transmission of NCC in or around a T. solium carrier” (Pawlowski, 2008). In contrast, taeniasis due to T. solium is not present in populations with no pork-eating. The crucial barrier is the religion “Prohibit pork-eating!” (Fig. 2).

Therefore, it is still sound that T/NCC due to T. solium may be common but cryptic NTDs in rural and remote areas where people eat pork under economic poor conditions. In contrast, people in developed countries with high income are basically safe for T. solium taeniasis, since they eat “safe pork” after meat inspection, and produced in “safe farms”. The most serious or cryptic NCC populations at risk are people in the border of the two societies where pork is or is not eaten, and people who are not infected with T. solium tapeworm(s). The main risk of NCC is through globalization with business, tourism and laborers from endemic areas. The border does not need to be geographic neighboring countries. T. solium tapeworm carriers contaminate other people in any societies by human to human infection with eggs through globalization (Fig. 2).

Cysticercosis is a highly disabilitating and even a mortal helminthic disease spreading worldwide, and causes similar disability-adjusted life years as dengue fever or African trypanosomiasis (Mathers et al., 2007, Fèbre et al., 2008, Zammarchi et al., 2013, Bobes et al., 2014). In conclusion, cysticercosis is one of the most neglected, neglected tropical diseases (NTDs) or neglected zoonotic diseases (NZDs) spreading all over the world (Budke et al., 2009).