Parasitology Lecture Notes PDF

Al-Turath University /College of Pharmacy Microbiology II-2nd semester/2nd year students (2023/2024) Assist. Prof. Dr. Shaymaa Abdalwahed Parasitology Lec 9 Blood and tissue flagellates Leishmania spp. Leishmania spp. caused Leishmaniasis disease spread by the bite of certain types of sandflies. Geo...

Al-Turath University /College of Pharmacy Microbiology II-2nd semester/2nd year students (2023/2024) Assist. Prof. Dr. Shaymaa Abdalwahed Parasitology Lec 9 Blood and tissue flagellates Leishmania spp. Leishmania spp. caused Leishmaniasis disease spread by the bite of certain types of sandflies. Geographical distribution divided the disease to: 1. Old world leishmaniasis caused by L. donovani, L. infantum, L. tropica, L. major, L. aethiopica are transmitted by the sandflies genus Phlebotomus; 2. New world leishmaniasis caused by L. mexicana, L. braziliensis and etc... are transmitted by the sandflies genus Lutzomyia and Psychodopygus. ✓ The term ‘New World’ refers to the Americas and ‘Old World’ is used for the rest of the world. Morphology and Life cycle of Leishmania The life cycle of Leishmania is completed in two hosts, humans and sandflies. Two stages are known; the amastigote which is spherical or subspherical and the promastigote which is pyriform or spindle shape with flagellum. Natural reservoir hosts humans, dogs and wild rodents. The adult female sandfly is a bloodsucker, usually feeding at night. When the fly bites a reservoir host or infected person with Leishmania, the pathogen reaches the stomach of the sandfly, the amastigotes quickly transform into elongated and motile forms called the promastigotes. 1 The promastigotes live extracellularly in the alimentary canal, reproducing asexually, then migrate to the proximal end of the gut. As the fly bites, the promastigotes are released from the proboscis (extensible tubular sucking organ from mouthpart) and introduced locally at the bite site. Once inside the human host, promastigotes invade macrophages. Inside the cells they transform back into the smaller amastigote form. The amastigotes replicate in the macrophage cell. After repeated multiplication, they break down their host cell by complete pressure of mass. The daughter cells protozoans then migrate to fresh cells (Cutaneous or mucocutaneous leishmaniasis) or through the bloodstream (visceral leishmaniasis) to find new hosts. In this way the infection is progressive, spreading to the host's mononuclear phagocyte system, particularly the spleen and liver. The free amastigotes in peripheral tissues are then ingested by sandfly to enter another cycle. Risk factors include poverty, malnutrition, deforestation, lack of sanitation and urbanization. Leishmaniasis is mainly a zoonotic disease. 1. Old world leishmaniasis A- Cutaneous leishmaniasis (oriental sore) Is the most common form caused by L. tropica, L. major, L. aethiopica, found in 88 tropical and subtropical countries which causes an open sore at the bite sites, which heals in a few months to a year and half, leaving an unpleasant looking scar. Diffuse cutaneous leishmaniasis produces widespread skin lesions which resemble leprosy, and may not heal on its own. Habitat is inside reticuloendothelial cells of the skin. 2 Pathogenesis: Leishmania invades human macrophages and replicates intracellularly. A raised, red lesion develops at the site of the bite (Incubation period varies from a few weeks to 6 months and in some cases it may be 1–2 years). Clinically, the lesion begins as a raised papule about 2.5 cm in diameter. The lesion then ulcerates and may become secondarily infected with bacteria. One factor restricting the parasites causing cutaneous leishmaniasis to the skin may simply be temperature. Diagnosis of Cutaneous leishmaniasis 1-A skin scraping with microscopic analysis using Wright or Giemsa stain. 2-Needle aspiration of tissue fluid from the margin of a lesion can yield fluid for culture (can be on NNN medium) to isolate the organism and identify the species. NNN medium is Novy-MacNeal-Nicolle (culture medium) 3-DNA testing (PCR). 4-Leishmanin skin test (Montenegro test) delayed-type hypersensitivity reaction to intradermal crude Leishmania antigen. In active visceral leishmaniasis, the test is almost always negative and a positive result usually appears 2–24 months after clinical recovery. Thus this test has no role in the diagnosis of the acute disease. B- Visceral leishmaniasis (kala-azar) Also known as black fever, and Dumdum fever, is the most severe form of leishmaniasis and, without proper diagnosis and treatment, is associated with high fatality. Caused by obligate intracellular parasite Leishmania donovani, L. infantum (infantile visceral leishmaniasis) Habitat: reticuloendothelial cells, predominately of liver, spleen, bone marrow and lymph nodes of man and dogs. 3 Pathogenesis: The parasite spreads from the site of inoculation to multiply in reticuloendothelial cells, especially in the liver, spleen, bone marrow and lymph nodes. This leads to progressive enlargement of these organs. The most typical symptoms are fever and the enlargement of the spleen, liver and lymph nodes, anaemia, leucopenia, and skin changes. Death is due to secondary infections. Sometime after successful treatment—generally a few months with African kala-azar, or as much as several years with the Indian strain—a secondary form of the disease may set in, called Post Kala-azar Dermal Leishmaniasis (PKDL). This condition caused by the reversal of L.donovani from viscerotropic to dermatotropic, manifests first as small, measle-like skin lesions on the face, which gradually increase in size and spread over the body. PKDL is not seen with L. infantum infection. Diagnosis of Visceral leishmaniasis (kala-azar) 1. Non specific tests: blood count, haemoglobin and serum protein estimation. 2. Parasitological diagnosis: blood film stained with Leishman or Giemsa stain, culture in specific media, visualization of the amastigotes in splenic or bone marrow aspirate, PCR (polymerase chain reaction) tests for the detection of Leishmania DNA. 3. Immunological tests: Serological testing is much more frequently used in areas where leishmaniasis is endemic. Leishmanin test is negative in active cases of kala-azar because the Cell mediated immunity is impaired in active kala-azar patients who consequently lack a delayed hypersensitivity response. Diagnosis of PKDL can be established by demonstration of amastigote form of L. donovani by a microscope of Leishman-stained smear prepared from the biopsy material obtained from nodular lesions. 4 2. New world leishmaniasis Mucocutaneous leishmaniasis (espundia) It causes both skin and mucosal ulcers with damage primarily of the nose and mouth (nasopharyngeal region). It can be highly disfiguring if not promptly treated. Caused by L. braziliensis and, L. mexicana, which are mainly found in certain South America, and Central America, respectively. Pathogenesis: Habitat of these parasites occur as intracellular parasites (amastigote form) inside the macrophages of the skin and mucous membrane of the nose and buccal cavity. Leishmania invades human macrophages and replicates intracellularly. A raised, red lesion develops at the site of the bite. The lesion may spontaneously heal with scarring, but then reappear elsewhere (especially as destructive mucocutaneous lesions). Diagnosis: as in cutaneous leishmaniasis Treatments: The treatment needed is determined by where the disease is acquired, the species of Leishmania, and the type of infection. Some possible medications used for visceral disease include liposomal amphotericin B, a combination of pentavalent antimonials and paromomycin, and miltefosine. For cutaneous disease, paromomycin, fluconazole, or pentamidine may be effective. Prevention Leishmaniasis can be partly prevented by: 1-sleeping under nets treated with insecticide. 2-spraying insecticides to kill sandflies. 3-treating people with the disease early to prevent further spread. 4-reservoir control programs. 5-Vaccination: leishmania is an intracellular pathogen since it requires strong cell mediated immunity to be controlled. However most of vaccines trials provide humoral response. 5 Blood and tissue flagellates Trypanosoma spp. Trypanosomes are extracellular protozoa, the name is derived from the Greek trypano- (borer) and soma (body) because of their corkscrew-like motion. Trypanosomiasis is a vector-born disease and is lethal without treatment. Kingdom: Protista. Phylum: Protozoa. Class: Mastigophora Taxonomy 1- The American and African trypanosomes: Trypanosoma brucei (T.b.) has 3 subspecies – Trypanosoma brucei brucei, T. b. gambiense and T. b. rhodesiense. The first one is an animal pathogen and the other two cause African trypanosomiasis or sleeping sickness in man. T. cruzi causes South American trypanosomiasis or Chagas’ disease. 2- Salivarian trypanosomes (Trypanosoma brucei and T. rhodesiense) are passed to the recipient in the saliva of the tsetse fly (Glossina spp.) and Stercorians trypanosomes (T. cruzi) are passed to the recipient in the feces of insects from the subfamily Triatominae bug (Reduviidae bug). African Trypanosomiasis (sleeping sickness) Life Cycle T. brucei completes its life cycle between tsetse fly (of the genus Glossina) and mammalian hosts, including humans, cattle, horses, and wild animals. In mammalian host: Infection occurs when a vector tsetse fly bites a mammalian host. The fly injects the metacyclic trypomastigotes into the skin tissue. The trypomastigotes enter the lymphatic system and into the bloodstream. The initial trypomastigotes are short and stumpy. Once inside the bloodstream, they grow into long and slender forms. Then, they multiply by binary fission. The daughter cells then become short and stumpy again. The long slender forms are able to penetrate the blood vessel endothelium and invade extravascular tissues, including the central nervous system (CNS). 6 Sometimes, wild animals can be infected by the tsetse fly and they act as reservoirs. In these animals, they do not produce the disease, but the live parasite can be transmitted back to the normal hosts. In tsetse fly: The short and stumpy trypomastigotes are taken up by tsetse fly during blood meal. The trypomastigotes enter the midgut of the fly where they become procyclic trypomastigotes. These rapidly divide to become epimastigotes. The epimastigotes migrate from the gut to the salivary glands and transform into short, stumpy trypomastigotes. These become the infective metacyclic trypomastigotes. They are injected into the mammalian host along with the saliva on biting. Complete development in the fly takes about 20 days. Only two subspecies, Trypanosoma brucei rhodesiense (Tbr) and Trypanosoma brucei gambiense (Tbg), have the additional feature of resistance to normal human serum (resist to active innate immune component of human serum and resist trypanosome lytic factor (TLF)). Although both subspecies are pathogenic to human, they differ significantly in virulence and geographical occurrence, T. b. gambiense causes chronic infections in West and Central Africa which can persist up to 10 years while T. b. rhodesiense is more prevalent in Eastern Africa and mostly results in acute human infections that can be lethal within a few months. 7 Both Tbr and Tbg causative agents of Human African Trypanosomiasis (sleeping sickness) in Africa. The development of protective vaccines has been unsuccessful until now, mainly due to the ability of African trypanosomes to escape adaptive immune responses by antigenic variation of the most abundant surface glycoprotein VSG (Variant-specific Surface Glycoprotein). Pathogenesis: In human infections, mortality results from neurological complications after penetration of the parasite into the central nervous system (CNS). Human African trypanosomiasis is characterized by two disease stages. During the first (haemolymphatic) stage of the infection, parasites will proliferate in the blood and the lymphatic circulation. Symptoms at this stage are nonspecific and include fever, lymphadenopathies, spleenomegaly and endrocrine disorders. Systemic inflammation finally if untreated, leads to increased blood-brain barrier (BBB) permeability allowing parasites to penetrate the CNS and cerebrospinal fluid (CSF), leading to the second (encephalitic) stage. The symptoms of this second stage include sensory, motoric and psychic disturbances, neuroendocrine abnormalities and disturbed circadian rhythms, eventually resulting in coma and death. The disturbed day-night cycles in the late stage of infection are characteristic for “sleeping sickness”. Diagnosis African Trypanosomiasis (sleeping sickness) The main points in diagnosis are the identification of the infecting trypanosome subspecies as well as the determination of the disease stage. To date, diagnosis of first stage mainly relies on microscopic detection of trypanosomes in blood smears and lymph node aspirates. Second stage diagnosis is based on parasite detection or lymphocyte counting in the CSF taken by lumbar puncture. Molecular and serological tools can be used for diagnosis. 8 Treatment African Trypanosomiasis (sleeping sickness) Treatment relies on suramine, pentamidine and melarsoprol. ✓ Severe side effects have often been reported, including anaphylactic shock, severe cutaneous reactions, neurotoxic signs and renal failure. However they does not cross the BBB, their application is limited to the treatment of the haemolymphatic stage. The generated nanobody (immunotherapeutic approach) was shown to be a promising tool for targeting effector molecules to the trypanosome membrane as it is able to penetrate into the VSG coat and bind to conserved trypanosome surface epitopes that are inaccessible to lager conventional antibodies. American Trypanosomiasis cause Chagas Disease Life Cycle The Trypanosoma cruzi (T.c) lives as trypomastigote in the blood and as an amastigote in reticuloendothelial cells and other tissue cells of man and many mammals. Life cycle starts in an animal reservoir, usually mammals, wild or domestic, including humans. A triatomine bug serves as the vector (vector-borne). While taking a blood meal, it ingests T. cruzi. A triatomine bug normally hide during the day and become active at night when they feed on animal and human blood. In the triatomine bug the parasite goes into the epimastigote stage, making it possible to reproduce. After reproducing through binary fission, the epimastigotes move onto the rectal cell wall (hindgut), where they become infectious. Infectious T. cruzi are called metacyclic trypomastigotes. When the triatomine bug subsequently takes a blood meal from a human, it defecates. The trypomastigotes enter the human host through the wound or by crossing mucous membranes or conjunctiva. Once in the vertebrate host, the metacyclic trypomastigotes, which is unable to replicate, must invade host cell within which it can differentiate into the replicating amastigote, which divides in the cytoplasm. After a number of rounds of replication by binary fission and after passing through promastigote and epimastigote forms, are again transformed into trypomastigote forms which are liberated in 9 the blood, which lyse the infected cell and escape to infect adjacent cells or disseminate throughout the body via the bloodstream and lymphatics. Trypomastigotes invade host cells and differentiate into amastigotes. Alternatively, they may be taken up by a triatomine insect during a blood meal and differentiate into epimastigotes in the insect midgut, thereby completing the life cycle. Life Cycle of T. cruzi Within the vertebrate host, parasites can infect any nucleated cell, but have a predilection for muscle, particularly of the heart and gastrointestinal tract. This tissue tropism ultimately leads to the two predominant clinical forms of chronic T. cruzi infection: cardiomyopathy and megacolon/megaesophagus. Pathogenesis of Chagas Disease (CD) Acute T. cruzi infection results from the contamination of wounds or mucous membranes with insect feces containing expelled infective parasites. Acute infection is marked by the development of localized swelling and erythema at the site of the insect bite, which is termed a chagoma. This is a result of the local replication of parasites and the influx of fluid and inflammatory cells into the infected area. 10 Infection through the conjunctiva can result in periorbital swelling, termed Romana’s sign. Lymphatic drainage of the infected area into regional lymph nodes results in activation and proliferation of cells, resulting in regional lymphadenopathy. As the process continues, the amastigotes transform into trypomastigotes, escape host cells and disseminate throughout the body. In chronic infection, tissue parasites are difficult to detect but significant interstitial fibrosis occurs, Cardiac involvement is signaled by the development of fibrosis within the heart muscle damaging the affected tissue. In the gastrointestinal tract, chronic infection leads to parasympathetic denervation, resulting in massive dilatation of the esophagus and/or colon. In later years these patients may experience the destruction of the nervous system and heart muscle, consequent cardiac arrhythmias or progressive heart failure, and sudden death. Diagnosis History of potential exposure to T. cruzi is important to document. 1-Microscopic examination for a blood film. 2- PCR. 3-Culture the blood on NNN (Novy-MCNeal-Nicolle) media. 4-Immunoassay. 5-Xenodiagnosis: Is a technique that uses the arthropod host as an indicator of infection. Uninfected reduviid bugs are allowed to feed on the blood of a patient who is suspected of having Chagas’ disease (T. cruzi infection). After 20–25 days, feces from the bugs are examined over a 3-month time frame for the presence of developmental stages of the parasite, which are found in the hindgut of the vector. This type of procedure is used primarily in South America for field work, and the appropriate bugs are raised in various laboratories specifically for this purpose. The term “xenodiagnosis” has also been applied to the diagnosis of trichinosis (Trichinella spiralis) result from roundworms (nematodes) from the genus Trichinella. Muscle tissue from a patient suspected of having the disease is fed to uninfected rats; the rats are then checked after the appropriate time for the presence of T. spiralis larvae, particularly in the diaphragm. 11 Treatment of Chagas Disease (CD) Nifurtimox and benznidazole. Both these agents suppress parasitemia and can cure acute phase of Chagas’disease in 60–80% of cases. Both medicines kill the parasite and are fully effective in curing the disease if given early in the acute phase, including in case of congenital transmission. Note: The mosquito/insect gut is divided into: Foregut, Midgut (midgut functions in storage and digestion of the bloodmeal) and Hindgut Thank You 12