Flummoxing flukes with PCR test

There are a variety of species of trematodes, more commonly known as flukes, which like to set up shop in humans. Many are tiny intestinal flukes, like the Heterophyidae and Lecithodendriidae families, which can cause gastrointestinal problems such as diarrhoea, vomiting and dyspepsia.

Others, such as liver flukes from the Opisthorchiidae families and blood flukes like the Schistosomatidae, are far more serious. Schistosomiasis, a chronic illness that has symptoms from mild anaemia to fibrosis of the internal organs, is considered the second most important parasitic disease after malaria and infects 200 million people worldwide.

Infections by Opisthochiidae are also widespread, particularly in Asia. Like the Schistosomatidae, the natural reservoir for Opisthochiidae is fresh water snails, with fish as second intermediate hosts. Humans pick up the parasites by swimming or bathing in infested water, or by eating raw or undercooked fish.

Digenetic trematodes like these species generally have a three-host lifecycle. The miracidium, or first larval stage of the fluke, penetrates the body wall of the snail host.

It then metamorphoses into a mother sporocyst, which gives rise to daughters, or rediae. These then give rise to cercariae, the free-swimming larval stage, which leave the snail host and penetrate or are swallowed by fish.

In the fish, the cercariae then develop into metacercariae and encase themselves in cysts, which are then consumed by humans and other animals if the fish is eaten raw or undercooked. The adults take up residence in the intestines or liver, where they reproduce, and their eggs are then passed on through faeces, starting the cycle over again.

In some parts of Asia, where eating raw fish is a common cultural practice, fluke infection is a common problem. In particular, two genera of Opisthorchiidae, Opisthorchis and Clonorchis, live in the human biliary passages, feeding off bile and causing a range of complaints.

These can range from general malaise and abdominal pain to gallstones and liver abscesses and, in cases of prolonged infection, cholangiocarcinoma, or cancer of the bile duct.

Clonorchis sinensis often causes cholelithiasis, commonly known as gallstones, while Opisthorchis viverrini is more strongly linked with cholangiocarcinoma. Endemic areas include Korea, China, Taiwan, Japan, Russia and northern Vietnam for C. sinensis, and Thailand, Laos, Cambodia and southern Vietnam for O. viverrini. Intestinal flukes are also widespread.

C. sinensis was not thought to be endemic in Thailand, but while working in a village in the central part of the country on developing a PCR test to distinguish between types of trematode species, the University of Queensland’s Dr Rebecca Traub and her colleagues have found that this is not necessarily so.

Their new PCR test is able to distinguish between O. viverrini, C. sinensis and other Opisthorchis-like eggs, including those from intestinal flukes. While the test will not necessarily be used on an everyday basis –it is hard to advocate a specific treatment for an unspecific parasite, but broad-spectrum anthelmintics are reasonably cheap and effective – it will be an advantage in epidemiological surveys and in control programs.

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Dog to human

Clonorchis has much lower host specificity than its relatives, so is happy to infect many other animals, meaning human-only treatment is not enough. But knowledge of other animals is where Rebecca Traub has an advantage.

Originally from Perth, Traub trained as a veterinary surgeon and worked in small animal practice for a few years before she heard the call of creatures both larger and much smaller than terriers and toms. Traub began a PhD at Murdoch University, specialising in gastro-intestinal parasites transmitted from dogs to humans.

She did fieldwork in Assam, a tea-growing area in northern India, where she developed molecular tools to distinguish species of hookworm directly from eggs in faeces. She was also the first to provide strong evidence from both an epidemiological and molecular level for the transmission of Giardia from dogs to humans.

Her PhD won her the John Frederick Adrian Sprent prize from the Australian Society for Parasitology and she later spent time at Cornell University in the US, looking at the molecular epidemiology of Giardia and Cryptosporidium in watersheds supplying New York City.

Her work on canine parasitic zoonoses, however, had caught the attention of Bayer Animal Health, which initially helped fund her PhD and later an ARC Linkage grant which brought her rushing back to Australia. She has continued the work at the University of Queensland, where she is now a lecturer in veterinary public health, and Bayer Animal Health continues to fund her work – currently Traub and PhD student Dr Megat Abd Rani are continuing the next chapter, investigating the veterinary and public health significance of canine vector-borne diseases in India.

Last year, Traub published a paper with Murdoch’s renowned parasitologist Professor Andrew Thompson on using PCR-based tools to pinpoint dogs as natural reservoirs of zoonotic ancylostomiasis, caused by the hookworm species Ancyclostoma ceylanicum.

This parasite can cause patent infections in humans – normally it takes up lodging in the skin of human feet and then dies off, but in some cases it continues up into the gastro-intestinal tract and causes non-specific gastrointestinal symptoms and may also cause anaemia.

While this research was concentrated on dog-loving temple communities in Thailand, one of Traub and Thompson’s former students, Dr Carly Palmer, found the hookworm in Australia. Following a four-year study of 2400 dogs and cats throughout the country, the researchers estimate that up to 10 per cent of hookworm-positive dogs here carry the worm, which they can pass on to humans.

“This is the huge advantage of using molecular methods,” Traub says. “It is only since the discovery of these tools that allow such specific amplification of pathogens from clinical samples that we are really getting a good idea of what’s present.

“The whole idea of looking at every single adult worm under the microscope … it’s just not possible to do large-scale surveys accurately.”

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Purge the purge

In the past, the only way to definitively identify the species of worm was to purge, an extraordinarily unpleasant experience for doctor, vet and patient, whether canine or human. More delicately known as expulsion chemotherapy, this process involves administering Epsom salts and a lot of water, much barfing and then the unpopular job of fishing through the end result looking for adult worms.

The alternative is microscopic examination of stools using smear tests like the Kato-Katz technique – long used for Schistosoma infection but not considered overly reliable – or sedimentation concentration using formalin-ethyl acetate or formalin-ether (FECT), to look for eggs.

These methods tell you if eggs are present but not what they are, as they all pretty much look the same. With eggs of similar morphologies, the most a microscopist can do is confirm eggs as ‘Opisthorchis or Clonorchis-like’.

Enter polymerase chain reaction, and the idea of amplifying specific sequences of DNA to discriminate between species. There are several PCR-based methods available, but even these have their limitations, Traub says. “The whole problem that I’ve experienced with previously published PCR tests is that they have not been really applied to the field,” she says.

One of the problems is that the primers cross-react with everything else in the sample. “There’s fungus and bacteria and all sorts of other parasites in there. When we tried to duplicate previously published PCRs on faecal samples positive for Opisthorchis-like eggs we often got about 15 different bands. It’s fine to say okay we have taken out eggs from flukes, and we have experimentally diluted them and used these primers and they amplify and they can be used in the field, but it’s not proof.

“The art is to get the primers and PCR conditions specific enough so that it is only going to target your fluke but also sensitive by targeting multi-copy genes that are semi-conserved.”

Traub’s methodology uses PCR-restriction fragment length polymorphism (RFLP), a quicker way of distinguishing the liver flukes by sequence and directly from eggs found in faeces. In developing the technique, the researchers homed in on a commonly used phylogenetic marker, the internal transcribed spacer (ITS-2) region of ribosomal DNA.

Traub, along with Thompson and colleagues from the University of Queensland, Thailand and Denmark, then conducted a study of a 5000-strong rural community in central Thailand, about 150 km from Bangkok. This community is in a rice-growing area with plenty of ponds full of snails and fish, and has a culture of eating the fish both raw and pickled.

Stool samples were collected from 335 people and tested using direct faecal smear, Kato-Katz and FECT. Samples were also fixed and sent to UQ for molecular testing and comparison.

At UQ, 31 faecal samples that were found microscopically to be positive for Opisthorchis-like eggs were PCRed, as were 30 samples identified as negative. Sequences of ITS-2 from several species were aligned and amplified.

Of the 31 samples identified as positive by microscopy, nine were actually negative on the PCR test. Of the 22 that were positive, PCR was able to distinguish that five were from C. sinensis, 14 from O. viverrini and three were didymozoids, intestinal flukes that infect fish but are usually mechanically passed by humans.

Of the 30 negative samples, seven were found to be positive, six for C. sinensis and one for O. viverrini.

While this study showed that the new PCR technique was more sensitive than other PCR tests and microscopic evaluation, one of the more interesting findings from the study was that it was the first report of a community endemic for C. sinensis in central Thailand.

The villagers reported only eating fish from local ponds, so it may be the case that C. sinensis is as widespread in Thailand as O. viverrini, Traub says.

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First intermediate hosts

This may put the focus back on the snails. The Parafossarulus and Bithynia species of snail are thought to act as first intermediate hosts of C. sinensis, and in Thailand are hosts for O. viverrini. Indeed, there are blood tests being developed to pick up Bithynia antigens to diagnose Opisthorchis infections.

Melanoides tuberculate, a known host for C. sinensis, has been found recently in Thailand, so this species may be the natural intermediate host for both, as it is in Vietnam.

“No one really knows because it is so difficult to identify the stages in the snail host,” Traub says. “Again, that requires digesting the entire snail and then looking at the stage involved, and then the same with the fish as well – digesting the tissue and looking at the metacercaria to identify what species it is.

“The PCR hopefully can be used to also screen intermediate host species and therefore give more information about the exact species of snail responsible. So in the long term this probably is a tool that will be utilised for baseline research purposes and will be very valuable in terms of control.”

In the meantime, Traub has plenty of other species on her plate. Traub’s PhD student Leigh Cuttell is attempting to prove that mainland Australia is Trichinella-free. Most people know Trichinella as an infection passed to humans through consuming undercooked pork, but it also infects reptiles like salt-water crocodiles and carnivorous birds. It is endemic in Tasmania and Papua New Guinea.

Failing to discover Trichinella on the Australian mainland will help justify Australia’s call to be considered Trichinella-free by the European Union, which currently enforces strict certification rules on all pork and wild game (feral pigs, horses, crocodiles) exported to Europe, costing the industry a significant sum in testing.

She is also collaborating with Dr Robyn Nagel at St Vincent’s Hospital in Toowoomba to study treatment failure for blastocystis, which is thought to infect about 10 per cent of the Australian population and has recently been linked to symptoms similar to irritable bowel syndrome.

Not widely known in the medical community, 50 per cent of treatments for the infection fail and Traub is trying to find out why.

Parasitic infection is ubiquitous and zoonoses widespread, so Traub suspects she’ll be in a job for some time. “As long as you are in veterinary public health, you are always employed.”