PHARMACEUTICAL MICROBIOLOGY - V. A. Galynkin - 2015

PART I. BIOLOGY OF MICROORGANISMS

CHAPTER 6. PROTOZOA

Protozoa — single-celled eukaryotic organisms featuring a significantly more complex functional Organization compared to Bacteria and Fungi. Their size ranges from 3 to 200 µm, with the largest testate amoebae reaching 2–3 cm in diameter.

Externally, the body of protozoa is covered by an elastic membrane known as the pellicle. In some species, The Cell membrane may incorporate supporting fibrils or even a mineral Skeleton. Their Organelles are identical to those found in other Eukaryotic Cells. Specific organelles of locomotion include pseudopodia, flagella, and cilia.

There are approximately 25,000 known species of protozoa, of which about 7,000 are pathogenic to plants, animals, and humans. Human-pathogenic species belong to 3 phyla: Sarcomastigophora, Apicomplexa, and Ciliophora (Fig. 42). The routes by which pathogenic protozoa enter The Human Body are similar to those of other pathogenic microorganisms.

Class="center">Fig. 42. Protozoa: 1 — Entamoeba histolytica, vegetative forms (a) and cysts (b); 2 — Leishmania donovani; 3 — Trypanosoma gambiense; 4 — Trichomonas vaginalis; 5 — Lamblia intestinalis; 6 — Plasmodium vivax in an erythrocyte, developmental stages: a) young form of the plasmodium, b) mature schizont, c) division form, d) gametocytes; 7. Balantidium coli.

6.1 Sporozoans

Sporozoans belong to the phylum Apicomplexa and the class Sporozoa, which consists exclusively of parasitic species. They are characterized by an exclusively sexual developmental pathway or an alternation of sexual and asexual cycles, typically associated with a change of hosts. Sporozoans derive their name from their ability to form specialized structures protected by a dense shell, conventionally referred to as spores. The greatest toll on human health is caused by malaria parasites and toxoplasmas, which infect up to 35% of the global population.

The genus Plasmodium comprises over 100 species parasitizing reptiles, birds, and animals. Four species are pathogenic to humans and cause malaria: Plasmodium vivax— the CAUSATIVE AGENT OF tertian malaria, P. malariae — quartan malaria, P. falciparum — falciparum (malignant tertian) malaria, and P. ovale — ovale malaria (a form similar to tertian malaria).

The life cycles of various plasmodium species are practically identical, comprising an asexual stage (schizogony) taking place in the human body, and a sexual stage (sporogony) within the vector — female mosquitoes of the genus Anopheles.

Sporogony occurs within the epithelial Cells of the mosquito's gut and lasts for 1–3 weeks. The process begins when male and female Gametes (gamonts) enter the mosquito along with the Blood of an infected host. The gamonts fuse in pairs to form zygotes, which penetrate the gut wall and develop into oocysts. The Contents of the oocysts undergo multiple divisions to produce sporozoites (spindle-shaped cells 11–15 µm in length) that disseminate throughout the insect's body. Some of these migrate to the mosquito's Salivary Glands, rendering it a disease vector.

Tissue schizogony of the plasmodium occurs in human hepatocytes and lasts for 1–2 weeks. Sporozoites invade Liver cells via the bloodstream within an hour after a mosquito bite. There, they divide to form merozoites (each sporozoite can generate between 2,000 and 40,000 merozoites), which rupture the hepatocytes and enter the bloodstream.

Erythrocytic schizogony takes place after merozoites invade erythrocytes, transforming into trophozoites (growing forms) measuring 2 µm; Microscopy of infected erythrocytes reveals resting forms containing a Nucleus with a single Chromatin granule, as well as forms with a pseudovacuole bearing a ring- or signet-ring-like appearance. The trophozoites subsequently enlarge and form multinucleated schizonts (dividing forms). The schizonts produce a new generation of merozoites that infect other erythrocytes. Each schizont can yield from 6 to 24 progeny merozoites. The release of merozoites from erythrocytes is accompanied by their destruction. Fever occurs at the exact moment merozoites are released from ruptured red Blood Cells. The development cycle lasts 72 hours for P. malariae and 48 hours for other species. Upon completion of the cycle, the Replication of P. malariae and P. falciparum in the liver ceases, whereas in P. vivax and P. ovale, a fraction of the sporozoites (hypnozoites) remain dormant in hepatocytes, forming latent foci that cause delayed relapses.

Inside certain erythrocytes, female and male gamonts develop, completing their maturation solely within the mosquito's body over a period of 7–45 days (depending on ambient Temperature).

Toxoplasma gondii — an intracellular parasite morphologically resembling an elongated orange segment, 4–7 µm in length, with one rounded end. Distributed worldwide, it causes Toxoplasmosis. Human infection occurs via the alimentary route through the ingestion of oocysts and tissue cysts (via undercooked meat products, unwashed vegetables, and fruits), through the Skin, and transplacentally. The infection rate among populations in various countries ranges from 4% to 68%; the pathogen has been isolated from virtually all mammals and many birds.

The life cycle includes stages of sexual and asexual reproduction. The primary and definitive hosts are domestic cats and other members of the feline family, within whose bodies the Sexual reproduction of the pathogen takes place. Primary infection of cats occurs upon ingestion of rodents containing oocysts, from which parasites emerge as sporozoites that invade intestinal cells and transform into trophozoites, multiplying by division (schizogony). Sexual reproduction also occurs within the cells of the intestinal mucosa. Merozoites resulting from schizogony transform into gametocytes. The fusion of oppositely sexed gametocytes leads to The formation of a zygote (oocyst). Oocysts are round structures with a dense shell, 9–14 µm in size, shed in feces and capable of surviving in the soil for extended periods.

The asexual reproduction cycle takes place within the human body. Sporozoites emerge from the oocysts and are actively engulfed by macrophages (abortive phagocytosis). Accompanied by macrophages, they disseminate through the Lymphatic vessels. Schizogony occurs within the macrophages; at a late stage, the macrophages perish, and the released parasites (tachyzoites) infect body cells (any nucleated cell is susceptible).

The majority of toxoplasmosis cases are asymptomatic; however, in immunocompromised individuals, the infection takes on a severe, predominantly fatal course.

The genus Sarcocystis is represented by coccidia closely related to toxoplasmas, which also utilize multiple hosts. Humans become infected by consuming insufficiently cooked beef or pork containing sarcocysts, which can lead to intestinal or muscular sarcocystosis.

The genus Babesia includes species pathogenic to animals and humans, causing babesiosis — a malaria-like disease particularly frequent in splenectomized patients.

The genus Cryptosporidium encompasses species parasitizing the intestinal epithelial cells of warm-blooded animals. Infection occurs via contaminated Water and food. In immunocompromised patients, they cause chronic gastrointestinal disorders.

6.2 Sarcodines

Sarcodines are classified under the phylum Sarcomastigophora, class Lobosea, order Amoebia. These are highly primitive organisms, mostly free-living, although some inhabit the intestines of humans and animals. Among pathogenic amoebae, Entamoeba histolytica is the most widespread.

The genus Entamoeba includes the only human-pathogenic species, E. histolytica, which causes amoebiasis (amoebic dysentery). The causative agent exists in several morphological forms.

The large vegetative form is a large cell measuring 20–60 µm. It differs from other amoebae by its jerky progressive movement, during which it forms pseudopodia. It is excreted in the feces during acute amoebiasis.

The tissue form is a small (20–25 µm) pathogenic form that invades the wall of the Large Intestine, leading to specific lesions.

The luminal form is the primary form of existence, which forms cysts.

Cysts are non-motile, spherical (8–15 µm), transparent formations that sometimes contain chromatoid bodies (aggregations of RNA and Proteins). When stained with Lugol's iodine, 4 ring-shaped nuclei become visible.

Life cycle. The definitive host is human. Luminal forms of the amoeba inhabit the upper section of the large intestine, feeding on bacteria and cellular detritus. Moving passively with the intestinal contents, they penetrate the distal PARTS OF THE gut and, under certain conditions (dehydration, disturbance of the microbial coenosis, changes in pH), form cysts.

From the intestine, cysts enter water, hands, and food (carried by flies) and invade the human body. In the Small Intestine, the cyst wall dissolves, and each nucleus divides, producing 8 daughter cells.

Amoebae penetrate the submucosa of the intestine, disrupting intercellular interactions, and produce a necrotoxin that destroys epithelial cells and causes necrosis of adjacent Tissues. The amoebae then invade Blood and Lymphatic vessels, through which they spread to other Organs.

Other pathogenic species of sarcodines are less common: Naegleria fowleri causes amoebic meningoencephalitis; amoebae of the genera Acanthamoeba and Hartmannella are causative agents of sporadic diseases manifested by necrotic lesions of the skin, cornea, and Internal Organs, most frequently in immunocompromised or debilitated individuals. They inhabit water and colonize air humidifier systems, which can lead to airborne transmission of the amoebae.

6.3 Flagellates

A defining feature of this class is the presence of flagella, which provide locomotion. In some species, this function is performed by an undulating membrane—a thin fold formed by the longitudinal connection of one of the flagella to the protozoan body. Flagellates include A large number of species parasitizing the human body, yet only some of them are recognized as pathogenic.

Trichomonas vaginalis has a pear-shaped body 14–30 µm long, an elongated nucleus displaced toward the anterior end, and vacuolated Cytoplasm. The anterior end bears 4 flagella and an undulating membrane that reaches the middle of the body. An axial filament—the axostyle—runs through the entire body, protruding at the posterior end as a small spine.

It causes Trichomoniasis, a sexually transmitted infection.

Other trichomonads also inhabit the human body: T. tenax, a commensal of the Oral Cavity, and T. hominis, a commensal of the large intestine.

Giardia lamblia (Lamblia intestinalis) has a pear-shaped body measuring 5–15 × 9–21 µm with a thickness of 2–4 µm; it exists as a vegetative form (trophozoite) and forms cysts.

Trophozoites possess 2 nuclei and 4 pairs of flagella located on the upper, lower, rear, and lateral surfaces. An adhesive disc surrounded by fibrils is located in the anterodorsal region for attachment to the epithelial cells. They absorb nutrients across their entire body surface. They reproduce by longitudinal fission and inhabit the upper sections of the small intestine.

Cysts are non-motile, oval, 10–14 µm long, contain 4 nuclei and an adhesive disc, and are excreted in the feces.

They cause giardiasis, which can manifest as latent parasitism or primarily as intestinal dysfunction.

The genus Leishmania. All species of this genus are Obligate Intracellular Parasites of mammals; in humans, certain species cause leishmaniasis. Four groups of causative agents are distinguished.

1. The L. tropica group — agents of Old World cutaneous leishmaniasis (Africa, Asia).

2. The L. mexicana group — agents of New World cutaneous and diffuse cutaneous leishmaniasis (Americas).

3. The L. brasiliensis group — agents of New World mucocutaneous leishmaniasis.

4. The L. donovani group — agents of Old World visceral leishmaniasis.

Life cycle. Leishmanias go through two developmental stages: amastigote (non-flagellated) and promastigote (flagellated). Flagellated forms (promastigotes) are motile and develop in the body of an insect vector (phlebotomine sandfly). The body is spindle-shaped, 10–20 µm long, and they reproduce by longitudinal fission.

Non-flagellated forms (amastigotes) parasitize mammalian cells. The cells are oval, 2–6 µm long, and reproduce by simple division.

The vectors of the disease are mosquitoes of the genera Phlebotomus and Lutzomyia, which become infected by taking blood meals from infected humans and animals. Within the first day, the ingested amastigotes transform into promastigotes in the mosquito's gut, multiply, and within 6-8 days accumulate in the Pharynx. Upon biting a human or animal, the pathogen invades skin cells or internal organs (depending on the Leishmania species), where the promastigotes transform back into amastigotes.

Genus Trypanosoma. All species are pathogenic to mammals, causing trypanosomiases in humans—infections that strongly resemble leishmaniases. The Life Cycle of the parasite takes place in both Human and Animal hosts, in whom trypanosomes cause severe and frequently fatal diseases.

The body of trypanosomes is elongated and narrow, featuring flagella and an undulating membrane. They reproduce exclusively asexually by longitudinal or multiple fission (schizogony).

Their developmental cycle involves polymorphism and alternation of hosts. In the gut of insect vectors, trypanosomes exist as epimastigotes—elongated cells with a flagellum originating near the anterior end and an indistinct undulating membrane. In the blood of humans and animals, they circulate as trypomastigotes—elongated cells with a flagellum originating at the posterior end and a clearly defined undulating membrane.

African and American trypanosomiases are distinguished. African trypanosomiases are caused by T. brucei (subspecies rhodosiense and gambiense). The vector is the tsetse fly (Glossina spp.). During a blood meal, trypomastigotes enter the fly's body, transform into epimastigotes, and multiply in the gut and salivary glands. After several weeks, progeny populations of the pathogen accumulate in the insect's body, enabling it to transmit trypanosomes to mammals.

The causative agent of American trypanososmiasis is T. cruzi, with vector assassin bugs of the genus Triatoma. The life cycle of T. cruzi resembles that of other trypanosomes; however, this species does not form trypomastigotes, and reproduction in the cells of warm-blooded hosts occurs in the amastigote form.

Dientamoeba fragilis was long considered a non-pathogenic amoeba, but was later classified as a flagellate. It causes diarrhea. In the human intestine, it exists in an amoeboid, non-flagellated form. Cysts have not been identified, but human-to-human transmission has been proven.

6.4 Ciliates

Among all Ciliate species inhabiting the human intestine, the only definitively pathogenic species is Balantidium coli, which causes balantidiasis (ciliary dysentery).

The vegetative form is a ciliated protozoan with an elongated, oval body measuring 30-100 x 30-150 µm. It feeds on bacteria, fungi, and other food particles ingested via a cytostome (cell Mouth) surrounded by cilia. The nuclear apparatus consists of a macronucleus and a micronucleus.

Cysts are spherical with a thin wall and can survive in the environment for several weeks. B. coli inhabits the intestines of pigs, in which it is mildly pathogenic. Cysts are shed in feces. Sources of infection include contaminated water and food.

Patients and carriers should not be regarded as sources of infection, since cyst formation in humans is rare, and infection via vegetative forms is virtually impossible.

Treatment of Protozoan Infections

Because protozoa are eukaryotic organisms, their METABOLISM closely resembles that of higher animals. This limits the number of targets for pharmacological intervention and renders protozoa insensitive to most antibacterial drugs. The capacity for developing resistance is even more pronounced in protozoa, particularly malaria plasmodia, than in bacteria.

The main drugs used to treat protozoan infections are listed in Table 18.

Table 18. Drugs for the Treatment of Protozoan Infections