Opisthorchiasis and CCA

Opisthorchiasis

(The liver flukes)

Classification

Phylum: Platyhelminths

Class: Trematoda

Order: Opisthorchida

Family: Opisthorchidae

Genus: Opisthorchis/Clonorchis/Metorchis

Medically Important strains:

Opisthorchis viverrini, O. felineus, Clonorchis sinensis, and Metorchis conjuctus

Morphology;

The parasite is leaf like elongate and lanceolate shape. Two testes, the testes and ovaries are on the posterior parts of the body. Uterus in the medium part and vitellin follicles form two bands along the lateral margins of the body. The excretory vesicle is Y shaped and eggs are small and contain miracidia.

It causes the diseases of gall bladder known as cholecystitis, cholangitis, cholelithiasis and cholengiocarcinoma, CCA. The diseases called Opisthorchiasis is kind of zoonotic food borne infection normally transmitted to human by consuming raw and uncooked snails and fishes.

Life cycle:

The opisthorchis spp needs two intermediate hosts to complete their life cycle. They must pass through the snails, Bithynia snails and fresh water fish mainly Cyprinid fishes,  before pass into the definitive hosts which are the fish-eating animals such as cats, dogs and even human. The definitive hosts pass the embryonated eggs to the feces. Eggs are ingested by the snails and the eggs are hatched into their cercariae. It has diameter of 154 x 75 micrometer covered with spines and possess elongated tail. These are free swimming larva form of Opisthorchis spp. These free swimming cercariae encyst into the skin of fish and changed into metacercaria, an infective form. It is double walled cyst surrounded by a thick layer of fish muscle, oval, size ranges from 201 x 167 micrometers. Piscivores definitive hosts including human when consume such raw and undercooked fishes, the metacercaria. The larval form then go on metamorphosis to form juvenile fluke in the upper part of intestine, duodenum. They migrate from intestine to gall bladder through the biliary duct. They develop into hermaphroditic adults and live for years in intra and extra hepatic bile ducts and gall bladder. They continuously lay eggs and pass through the feces. The life cycle repeats again.

Figure 1: Life cycle of Opisthorchis spp.

Mode of Transmission:

Faecal oral route

By eating raw or improperly cooked fish dishes and poor sanitation and hygiene.

Reservoir of hosts

·         Good habitat for the intermediate hosts and presence of reservoir hosts. (Rice paddy field, water logged fields, fresh water ponds and stream where Pisces and snails coexist.

·         Definitive hosts like cats and dogs (Human are also definitive hosts so carriers and infected human are also three reservoirs).

Pathogenesis

The pathogenesis of Opisthorchis viverrini depends on the survival of worm in the human biliary duct, a hostile environment, mechanical damage to the liver and gall bladder, host immune response to the infection and metabolic products of worms inducing cell transformation and proliferation leading to cholangiocarcinoma, CAA to the host. The main histopathological features of Opisthorchiasis in both animals and human are epithelial desquamation, epithelial adenomatous, hyperplasia, goblet cell metaplasia, inflammation, periductal fibrosis and granuloma formation.

1.       Survival in the hostile environment of human biliary duct

The first adaptation the worm needs to cause infection to human is to adjust into the hostile environment of human biliary duct. The newly excysted juvenile worm of Opisthorchis viverrini use glycogen granules and lipid droplet stored in their excretory granules for energy source for migration. Once they reach to the biliary duct the glycogen granules get scared and so they start to use the large amounts of lipoprotein metabolites present there including HDL, LDL, IDL, and VLDL. These lipoproteins are composed of cholesterol, phospholipids, triglycerides as well as few amphipathic proteins. They soon develop into mature adults.  The environment in the biliary duct is hostile for parasite as there is low oxygen tension, strong oxidizing agents like singlet oxygen is produced there and the liver cells also have to frequently expose to the toxic metabolic products and carcinogens as well, However, O. viverrini has developed few mechanisms to adopt there. They produce SODs and glutathione S. transferases to neutralize the toxic oxygen derivatives like nascent oxygen, xenobiotics etc.

2.       Mechanical injury:

The adult worm easily survives and damage the biliary tissues. The oral and ventral suckers can hook to the biliary epithelium and cause epithelium desquamation in the early infections. As the flukes grow up the lesions are more pronounce and lead to ulcer. Their eggs are entrapped in the periductal tissue through the ulcers causing granulomatous inflammation. These can cause the partial obstruction, bile stasis, increase biliary pressure and enhance more entrapment forming granuloma. Such granuloma are the major cause of obstructive jaundice and cholangiocarcinoma, CCA).

3.       Metabolic products of worms induce the cell transformation

As previously described the flukes use many kinds of lipoproteins and proteins in the biliary duct and release various toxic biproducts from the tegument, known as excretory-secretory products (ES products). These biproducts not only contain the proteases for the digestion of tissues causing epithelial adenomatous, but also contain toxics substances which act as mitogen. This induces hyperplasia, metaplasia, dysplasia of biliary cells and leads to development of cholangiocarcinoma CCA, final complication of Opisthorchiasis. They also are responsible to cell cytotoxicity, cell injury and apoptosis. One of the biomarkers of this infections, 5-bromodeoxyuridine can be easily detected in the biliary secretions.

CCA development

Chronic infection of O. viverrini is a major risk factor for CCA development. Multifactorial aetiology with genetic alterations in tumor suppressor genes as well as proto-oncogenes are involved in CCA development. Few predisposing factors are;

·         Exogenous carsinogens-nitrosamines from fermented fishes

·         Fibrosis linked to metastasis associated protein 1 and interleukin 6

·         Cell damage and death due to Ov feeding

·         Transforming growth factor betta -mediated cell proliferation to ES molecules

Damage to biliary epithelium (interface between bile duct and hepatocytes) lead to chronic exposure of cholagiocytes to conjugated bile acids that can induce the expression of inflammatory mediators (IL-6 and IL-8) leading to increased oxidative stress and DNA damage and subsequent fibrosis and tumorigenesis. The secretion of mitogenic granulin like growth factor (Ov GRN-1) that directly modulate host cell proliferation.

Signs and Symptoms:

Most of infections are asymptomatic while those of symptomatic patients show some common symptoms like diarrhea, abdominal pain; epigastric and right upper quadrant pain with hot sensation. Fever, anorexia, nausea, loss of weight, poor appetite and also common. 

The patients may also have edema, icterus and eosinophilia. The ascites is also present. Obstructive jaundice and hepatomegaly occur in chronic infections. Moreover, the chronicity is characterized by cholecystitis, cholangitis, cholelithiasis and cholangiocarcinoma.

Immune response

Innate immunity: macrophages, eosinophils, monocytes, mast cells and lymphocytes

Humoral Immunity: IgM, IgG, IgA, and IgE

Cell mediated immunity: Cytokines, interleukin (IL-4), growth factors (TGF-beta) and IL-10. TGF-beta and IL-10 have a role in blocking effective immunity.

Laboratory Diagnosis:

Microscopy

Immunodiagnosis:

Antigen preparations: crude extracts of adult worms, metacercaria, eggs and intermediate hosts, ES products of adult worms, partially purified fractions of worms and eggs

Monoclonal Ab-purified Ov oral antigen (highly effective 100% sensitive and specific)

Copro antigens for the epidemiological study

Stool in TCA/mAb-ELISA

97.9% sensitive, 54.2% specific

Tegument serum/ELISA

100% sensitive and 94.9% specific

Molecular Diagnosis:

For O. viverrini and C. sinensis

·         Satellite DNA, ITS1, ITS2 and mitochondrial DNA (apply for conventional and real time PCR)

·         The retrotransposon of O. viverrini (new alternative marker)

PCR sensitivity can be improved by using cetyltrimethylammonium bromide (CTAB) during DNA preparation to remove PCR inhibitor.

Loop mediated isothermal amplification (LAMP)

For the species-specific PCR, genetic markers such as mitochondrial genes, ribosomal ITS1 and ITS2 can be used in different molecular tools such as PCR-RFLP, multiplex PCR, real time PCR, multiplex ligation-depended probe amplification MLPA and pyrosequencing.

Treatment

Praziquantel 25 mg/Kg/Day 3 times a day

40 mg/kg once

Problems for the control of Opisthorchiasis infections

·         Some of the traditional foods which are very popular in different countries. The traditional foods are made from fishes and normally not cooked or improperly cooked.

·         Metacercariae of O. viverrini were eliminated in a high salt concentration recipe of 3 day fermented crosswise cut streaks of cyprinid fish and also in fish grilled at 80C for 12 min.

·         Acid fermentation as in stick rice fish fermentation can destroy nearly all viable metacercariae after 69 h.

·         One of the potential hazards in cold smoked fish: Freezing raw fish prior to smoking remains the most effective way to ensure that viable parasites are not present in clod smoked products consumed by the public.

Phylogenetic analysis:

What significance do the helminthes species complexes have for the prevention, diagnosis and treatment of human infections?

Knowledge that some species within the O. viverrini complex are more pathogenic than others would allow screening to be concentrated in the areas where these species are present.

From the medical point of view, there are numerous potential problems

·         Are there differences in virulence within the species complex?
·         Does molecular diagnosis work for all species?
·         Is the susceptibility to drugs the same?
·         Has resistance to drugs developed in some species but not others?
·         Which hosts must be considered in control programs?
·         What should be explained in education programs?
·         What levels of morbidity and mortality are associated with each cryptic species?

In order to answer the above questions, we must know about the genetic diversity of O. viverrini and we need to study about the phylogenetic analysis of each species.