Entamoeba histolytica, Entamoeba dispar and E. moshkovskii - IgG antibodies; Molecular diagnosis (PCR); Species identification (PCR and sequencing)
When we speak of amoeba related to infections or human colonization we refer to species belonging to the genera Entamoeba, Endolimax and Iodamoeba, included in the family Entamoebidae. Among the species of this family Entamoeba histolytica is the only intestinal amoeba of recognized pathogenicity, responsible for amoebic dysentery, a disease that affects 40-50 million people a year and accounts for around 100,000 deaths per year.
The remaining intestinal amoeba species, E. dispar, E. moshkovskii, E. hartmanni, E. coli, E. polecki, Endolimax nana and Iodamoeba buetschlii, are considered nonpathogenic. However, they are able to colonize human intestines. The most frequent are E. histolytica and E. dispar. It is estimated that both infect approximately 12% of the world's population.
In the second half of the 20th century, there has been much discussion about the criteria for differentiating the E. histolytica and E. dispar species and knowing their relative pathogenicity. After a decade of debate, the biochemical, immunological and genetic differences between E. histolytica and E. dispar, previously known as pathogenic and nonpathogenic, respectively, have been shown to be sufficient to formally differentiate them as two separate species. However, the impossibility of being able to establish clear microscopic morphological differences between both species has motivated that both are analytically included under the binomial "Entamoeba complex". More recently, another species, E. moshkovskii, has been added to this complex, whose first isolations were in wastewater from several countries, but in recent years its presence in human faeces has been reported.
In this way, the "Entamoeba complex" would come to encompass E. histolytica, of recognized pathogenicity, and E. dispar and E. moshkovskii, considered non-pathogenic.
Among the mechanisms of pathogenicity in Entamoeba histolytica is the presence of the galactose-galactosamine lectin on the surface of the trophozoites, responsible for adhesion to intestinal cells. Also have been identified soluble polypeptides called ameboporos, which are inserted into the membrane of the target cell and induce cell lysis. In addition, cysteine proteases have been characterized, capable of degrading different components of the extracellular matrix. Cysteine proteases are also involved in the evasion of the immune response as they degrade IgA and IgG immunoglobulins, and C3a and C5a anaphylatoxins. In E. dispar the presence of ameboporos and cysteine proteases has been demonstrated in lower concentration and with lower biological activity that would rule out its pathogenicity. However, other recent studies, in vitro and in vivo, have offered evidence that some strains of E. dispar of different origins are capable of producing liver damage and destroying cell lines of culture.
The main reservoir of these amoebae is the human being itself, in which the protozoan manifests itself in two evolutionary forms during the life cycle: cyst and trophozoite.
The infection is caused by the ingestion of mature cyst resistant to gastric juices. Once it reaches the small intestine, a process in which the cyst releases a tretranucleated amoeba that, by nuclear multiplication, originates an 8-nucleus amoeba. Subsequently, cytoplasm fragmentation occurs in 8 small amoebas (metacystic) that are transformed into the corresponding trophozoites, responsible for the colonization of the colon where they feed on bacteria and cellular detritus. In this phase the generation of new cysts and more trophozoites takes place, which can sometimes invade other organs and produce abscesses at distance (hepatic or other less frequent locations). The cysts end being eliminated along with the feces, and their viability is of weeks to months, time in which they can cause new infections if they are ingested.
This group of intestinal amoebas shares a series of common epidemiological characteristics such as their cosmopolitan distribution, and they have the same mechanism of transmission, always linked to the ingestion of mature and infectious cysts through a fecal-oral transmission.
The actual prevalence of this group of amoebae is not always known, in some cases due to the degree of expertise at the time of establishing the specific diagnosis and in others for not finding out as they are not considered pathogenic. On the other hand, the epidemiology of the integrating species of the "Entamoeba complex" remains uncertain because most of the existing data has been obtained through methods unable to distinguish morphologically the 3 species: E. histolytica, E. dispar and E. moshkovskii.
In fact, recent molecular studies indicate that E. dispar is 10 times more frequent than E. histolytica, being the cause of 90% of human infections by species of the "Entamoeba complex". The majority of asymptomatic people infected by one of these species are colonized by E. dispar. However, in some infections the prevalence of E. histolytica in asymptomatic individuals has increased. E. moshkovskii has been found in human feces in North America, Italy, South Africa, Bangladesh, India, Thailand, Australia, Turkey and Iran, with figures up to 20% of parasitization in children, even in mixed infections with the other species of the complex.
Of the remaining species, and leaving aside E. polecki, which in Papua New Guinea reaches a human prevalence of 30%, very associated with contact with pigs, the prevalences vary depending on whether the studies are carried out in developed countries or not, ages of study or whether the population is symptomatic or not.
Another epidemiological characteristic common to the group is that they are considered non-pathogenic species. Without doubt there is consensus on the consideration of being species not associated with disease, which does not exclude that in the literature there is some evidence pointing in another direction. For example, gastrointestinal disorders have been reported in patients infected with E. polecki and with E. dispar, and even with mixed infection with E. dispar and E. moshkovskii. In this sense, it is known that E. dispar can produce in vitro intestinal lesions of varying intensity, even leading to destruction of the intestinal epithelium. In addition, there is evidence of pathological changes in some humans after infection with this species.
Although all of these amoebas are considered nonpathogenic, it should not be strange to think that the occurrence of them in the gastrointestinal tract could predispose to infection with other enteropathogens, modulate the immune response and thus facilitate secondary infections and even different degrees of mutiparasitism infection.
The diagnosis of amoebic infections has been based for a long time on the microscopic examination of the stool. However, deficiencies of this technique have been reported, especially due to the impossibility of differentiating with sufficient sensitivity and specificity the different species of the Entamoeba complex. In fact, the cyst and trophozoite forms of E. histolytica and E. dispar are morphologically indistinguishable in light microscopy.
Specific techniques, such as the cultivation of trophozoites by the Sargeaunt method and the typing of isozymes by the joint analysis of the electrophoretic profile of several isoenzymes, make it possible to differentiate E. histolytica and E. dispar. However, these techniques are very laborious and are not applicable in the usual diagnosis. The search for parasitic coproantigens by immunoenzymatic techniques (ELISA) often presents cross-reactivity between the two species. Serological techniques for diagnosis have a very limited use because they require detecting a seroconversion and their inability to distinguish an old infection from a recent infection in highly endemic areas.
The new approaches for the identification of these two species are based on molecular methods such as the polymerase chain reaction (PCR), which provides greater sensitivity and is highly specific since it is studied directly to the DNA of E. histolytica or E. dispar. The nested multiple PCR (NM-PCR) performed from fecal and liver abscess aspirate samples, directed to the 18S ribosomal gene followed by a digestion with endonucleases has been evaluated for the diagnosis of amebiasis.
Today, the WHO recommends a reassessment of the epidemiology of amebiasis using molecular techniques for the detection of E. histolytica, and to differentiate E. histolytica and E. dispar, important in deciding on the treatment of possible cases.
Tests carried out in IVAMI:
- Molecular diagnosis by amplification of the 18S ribosomal gene and subsequent sequencing to identify the intestinal amoeba species.
- IgG antibodies.
- Stool samples.
- Aspirate of intestinal and liver abscesses or other location.
- Serum for IgG antibodies.
Conservation and shipment of the sample:
- Refrigerated (preferred) for less than 2 days, or frozen if the storage period is longer.
Deadline for delivery of results:
- Molecular diagnosis of the DNA of the intestinal amoeba species (PCR): 24 hours.
- Identification of the intestinal amoeba species by sequencing the coding region of the 18S ribosomal gene: 48 to 72 hours.
- IgG antibodies: 72 hours.
Cost of the test:
- Molecular diagnosis (PCR): Consult firstname.lastname@example.org
- Identification by sequencing of the coding region of the 18S ribosomal gene: Consult email@example.com
- IgG antibodies: Consult firstname.lastname@example.org.