3.2 Amoebiasis and intestinal flora

Two species, which are structurally equal but genetically separated, must be distinguished: the facultative pathogen Entamoeba histolytica sensu stricto (Schaudinn 1903, Walker 1911), which, in 9 of 10 cases causes no symptoms, and the nearly exclusively commensal E. dispar (Brumpt 1912). No interspecific exchange of genes occurs between the two species but evidently an intraspecific exchange, i.e. within each of the two species, takes place.

Results of biochemical, immunological and genetic investigations of isolates from patients with and without invasive amoebiasis (see below) indicate that the two species are distributed at different frequencies that are statistically significant. However, no gene for pathogenicity has been detected. On the contrary, by changing the culture conditions in vitro, a stock obtained from a non-pathogenic primary isolate can be shifted to a strain that is toxic in vitro for cells and a pathogen in animal experiments. Thereafter, the strain can be reversed to a non-pathogenic form.

Life cycle: After the ingestion of cysts, single-nucleated minuta-forms (10-20 μm) hatch in the chymus of the small intestine. These trophozoites gain energy for growth by the glycolysis of carbohydrates. Membrane lipids and proteins are obtained from exogenous sources: several hours after phagocytosis, the bacterial enzymes are still active and have to be renewed continously. Finally, the trophozoites encyst and form chromidial bodies; the nuclei divide twice, each time becoming smaller. The excreted cyst (10-15 μm) becomes infectious after contact with CO2. Infection remains symptomatically non-apparent and the parasite-host-relationship is commensal (fig. 3.8 above, page 192).

In cases of invasive intestinal infection, the amoebic trophozoites interfere with the feed-back mechanisms exerted between the bacterial microflora living inside the mucus (viscous film covering the epithelium) and the Paneth cells in the crypts of Lieberkühn. The trophozoites turn into magna-forms (20-30 μm), which show a clear ectoplasm separated from the inner endoplasm, the latter mostly with internalized red blood cells (RBCs). However, mitochondria, peroxysomae, rough endoplasmic reticulum or Golgi dictyosomae are absent. After adherence to the surface of the mucus and degradation of the glycoproteins, the magna-forms adhere to the gut epithelium: Cystein proteases cause apoptosis and necrosis of epithelium cells accompanied by punctual colitis. For the penetration of the epithelium, antibodies are depolarized and degraded and complement is inactivated. Chemokines of the amoebae mobilize macrophages and neutrophilic leucocytes (fig. 3.9, page 193). They cause intestinal ulcers, which may expand into the muscularis and break through to the bowel cavity. Magna-forms transported via the portal vein to the liver turn into tissue forms that generate cavernae by the lysis of tissue and feed on the fragments of cells. Tissue and magna-forms never encyst (fig. 3.8 below, page 192).

The many small ulcers at the inner surface of the colon give rise to mucus with haemoglobin from lysed RBCs, which can be observe as superficial layers on deposited stools. The stools are paste-like to mushy with a slight tendency to diarrhoea, i.e. the loss of water is physiologically negligible. In cases of a necrotic hole (caverna), for example in the liver, the lesion is called an amoebic abscess and remains bacteriologically sterile. The parasitic infection is designated as an extra-intestinal amoebiasis. At this stage, all the other intestinal stages of the amoebae may be missed.

Magna and tissue forms induce strong immune responses but no protective immunity; without re-infection, intestinal lesions heal spontaneously. The change from the commensal to the pathogenic form is based on the effectivity of the amoeba proteins, i.e. involves a change from the endocytic lysis of bacteria to the exocytic lysis of intestinal epithelium cells. The latter is simulated in vitro by granulocytes (fig. 3.10, page 197). In vivo, this is apparently not explained by monocausal events.

In addition to microscopic diagnosis on untreated samples or samples stained with iodine-iodine-potassium or fixed with iron-haematoxylin, enzyme-linked immunosorbent analysis (ELISA) of faeces can be used to establish the presence of a relevant antigen, or the polymerase chain reaction (PCR) can be employed to reveal relevant DNA sequences. E. histolytica s.s. is seldom found together with E. dispar; however, the latter is mostly associated with 2-3 intestinal protozoan types (commensal amoebae, lambliae, ciliates).

For the chemotherapy of all clinical forms, derivatives of nitroimidazole are applied orally. An abscess is cured solely by infusion. Abdominal typhus and paratyphus and bacterial dysentery can be distinguished from an amoebiasis by the clinical aspects of the patient and by the characteristics of the faeces (Box 3.1, page 194).

In man, in addition to E. histolytica s.s. and E. dispar, three other commensal amoebae are observed with various frequencies: Entamoeba coli (cyst up to 8 nuclei) and E. hartmanni (trophozoit 4-10 μm, cyst 7-10 μm) in the colon, E. gingivalis in the mouth cavity.

E. histolytica s.s. is a balance strategist (chapter 8.1.2 and fig. 8.1, page 282): The course of the apparent disease is chronic and heals spontaneously. Lethality is rare and concerns predominantly the older age classes. The basic cycle of cyst – trophozoite – cyst guarantees the survival of the exclusively commensal stages in the chymus, which is rich in bacteria, without intervention of the immune apparatus. There is obviously no selective pressure in favour of or against a parasitic intestinal or extra-intestinal invasion. Invasive amoebiasis does not support transmission; on the contrary, it endangers the survival of the pathogen together with its host. The netto case reproduction rate of the non-apparent, solely transferring amoebiasis is as a rule super-optimal, even at low prevalences.

These findings can only be understood biologically, taking into consideration the conditions in the human colon. First, they cannot be simulated completely in vitro. Second, the isolation and culture of amoebae involve several selective processes; therefore, a strain reared in vitro is different from the original isolate. No completely chemically defined culture medium that is absolutely free of bacteria is available. The strictly commensal amoeba contribute to the stabilisation of the intestinal flora. E. histolytica sensu lato has been known to overcome infection by Shigellae in the early stages. The transition to parasitism occurs at a super-optimal case reproduction rate. Amoebae of free-living vertebrate hosts exhibit a cyclic transmission pattern between predator and prey, thus stabilising the corresponding ecosystem.

The epidemiology of human amoebiasis depends on hygiene with respect to drinking water and food. During the last two centuries, this parasitic disease has been restricted to tropical and subtropical regions in which the high prevalences, which may comprise as much as half of a population, , are attributable to the scarcity of clean drinking water in arid zones and to the prolonged infectivity of the externalised cysts in humid-temperate regions. In affected patients, the rate of invasive intestinal and extra-intestinal amoebiasis varies according to the geographical region: In Asia (India), it is many times higher then in all other continents. E. histolytica s.s. and E. dispar are specific for primates, the disease is an anthroponosis (table 3.2, page 195).

The genus Entamoeba occurs in all terrestrial vertebrates, including amphibians and reptiles: E. cuniculi in the rabbit (cyst 8 nuclei), E, cobaye (cyst 8 nuclei), E. caviae in the guinea pig, E. gallinarum and E. janisae in chicken birds (Galliformes), E. bovis in cattle, E. equis in horse. E. invadens in reptiles (lethal in snakes, commensal in turtles). E. moshkovskii is free-living and is not pathogenic, even under experimental conditions.

The conditions in the human colon are almost anaerobic and resorbed water is replaced by the secretion of potassium and bicarbonate. The microbes of the gastero-intestinal tract establish an ecosystem comprising more than 400 species of bactera. The upper part, viz. the stomach, duodenum, jejunum and upper ileum, contains a sparce microflora that originates from the mouth cavity and pharynx but that is decimated by gastric acid to approximately 10 organisms per ml. Peristalsis prevents reflux. The colon contains a luxurious microflora of 1011 bactera per gram, which characteristically distribute lengthwise along the colon and over its cross-section. They enzymatically decouple substances that have been previously conjugated in the liver and that pour into the chymus together with the bile. Once resorbed, they arrive in the liver via the portal vain: Entero-hepatic circulation. The microflora enables the resorption of vitamins and digests roughage for energy. The flora has a protective capacity; at imbalance, potentially pathogenic germs may propagate excessively and favour invasive amoebiasis, as established in an human self-experiment. After death, the gastrointestinal tract decomposes first.

The incrimination of intestinal amoeba as a pathogen by using the postulates of Henle-Koch is impossible (Box 3.2, page 199). Culture in vitro begins with a polyxenic isolate, which has to be transferred from anaerobic to aerobic monoxenic conditions, thereafter being returned to anaerobic conditions. By the application of antibiotics, fewer than 105 bactera per ml can be achieved and, finally, the amoebae are fed on lethally irradiated bactera. The persistence of pathogenicity has to be established by monolayered tissue cultures or granulocytes and in an experiment in vivo with animals.

Rare cases of.rapid lethal amoebic attack in man have recently become known and affect the central nervous system post mortem. The neurotropic pathogens are thermophil and amphizoic. An exozoic phase in the mud of rivers or in humid ground may transform into an enzooic phase and invade ganglia and the cerebrospinal fluid. Naegleria spec. carries two flagella in its exozoic form; once inoculated into mice, it attacks the brain. Acanthamoeba spec., named because of its tapering pseudopodia, attacks the previously damaged cornea of the eye and thereafter the central nervous system. All these amoebae are easily to cultivate and occur ubiquitously in fresh and saline water, in soil and in dust in the air and have been isolated from drinking water containers, air-conditioners, cooling water of nuclear power stations and hot-water tanks.

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