BMS201 Blood Parasites Lab Fall 2024 PDF

BMS201 Practical Lab No: 1 Title: Plasmodium spp. Babesia spp.- Polymorphic and Monomorphic Trypanosomes Prof. Khalifa E. Khalifa Medicine and Surgery Program Spring 2024 By the end of this lecture, you sh...

BMS201 Practical Lab No: 1 Title: Plasmodium spp. Babesia spp.- Polymorphic and Monomorphic Trypanosomes Prof. Khalifa E. Khalifa Medicine and Surgery Program Spring 2024 By the end of this lecture, you should be able to: 1. Identify diagnostic stages of Plasmodium vivax and P. falciparum form their morphologic characteristics 2. Identify the diagnostic stages of Babesia spp. and differentiate it from P. falciparum in blood film. 3. Identify the vector of Babesia spp. and describe the type of development inside vector 4. Discuss the mode of transmission of Babesia spp. other than vector bite 5. Identify the infective and diagnostic stages of polymorphic and monomorphic trypanosomes from their morphologic characteristics. 6. Identify the vectors of polymorphic and monomorphic trypanosomes and describe the type of development inside vectors. Blood, Lymphatics and Macrophage Phagocytic System Parasites Class Parasite System Helminths (Nematodes) Wuchereria bancrofti Lymphatic system Brugia malayi Protozoa (Hemoflagellates) Leishmania donovani Macrophage Phagocytic system of blood and viscera Protozoa (Hemofalgellates) Trypanosoma brucei spp. Blood outside RBCs and Lymphatics Trypanosoma cruzi Blood outside RBCs and intracellularly in tissue of mesenchymal origin) Protozoa (Apicomplexa) Plasmodium spp. Blood inside RBCs Babesia spp. Protozoa (Apicomplexa) Toxoplasma gondii Macrophage Phagocytic system and any nucleated cell Plasmodium spp. “Malaria”  Plasmodium spp. Disease: Malaria P. vivax P. ovale P. falciparum P. malariae 10/6/2024  IH DH Sporogony Infective stage: Sporozoites (bite of Female Anopheles ), Exoerythrocytic Blood stages (blood schizogny transfusion) Mode of infection: Bite of infected female Anopheles, Blood transfusion, Congenital Gametogony Use of contaminated syringes Mode of transmission: Cyclopropagtaive Erythrocytic schizogny 10/6/2024  Blood (Diagnostic) Stages of Plasmodium vivax Early trophozoite (Ring form) Ring form with single chromatin dot, thin rim of cytoplasm and a vacuole. Size: 1/3 of RBC. Late trophozoite (amoeboid form) irregular with amoeboid cytoplasm. Mature schizont With 12-24 merozoites, clumped malaria pigment. Size: fills RBC. Male (micro) gametocyte Rounded with diffuse chromatin. Size: fills RBC. Female (macro) gametocyte Rounded with compact chromatin. Size: fills RBC. Red cell: enlarged. Stippling: Schuffner's dots.  Blood (Diagnostic) Stages Plasmodium falciparum Early trophozoite (Ring form) Ring form with single chromatin dot, very thin rim of cytoplasm and a vacuole. Usually 2 chromatin dots, multiple infections (more than one ring) and accolé (marginal) forms. Size: 1/6 of RBC. Late trophozoite with compact cytoplasm. Mature schizont With 12-24 merozoites, clumped malaria pigment. Size: 2/3 RBC. Male (micro) gametocyte Crescent with diffuse chromatin. Size: larger than RBC. Female (macro) gametocyte Crescent with compact chromatin. Size: larger than RBC. Red cell: unchanged. Stippling: Maurer's clefts. Rapid Malaria Diagnostic tests (ICT- Antigen detection) QBC Screening of blood donors for ant- Plasmodium antibodies by ELISA Plasmodium vivax diagnostic stages Schizont stage Trophozoite stage Ring stage Gametocyte stage Plasmodium vivax Ring stage 1 1) Thin loop of cytoplasm. 2) Single chromatin mass. 3) It occupies 1/3 of RBC. 2 4) Single infection of RBC is usual. 5) Enlarged RBC. Plasmodium vivax Trophozoite stage Shape: irregular outline. Chromatin: single dot. Stippling: yellowish brown scattered very fine particles (Schüffner’s dots). Infected RBC: enlarged. Schuffner’s dots Trophozoite stage of Plasmodium vivax 10/6/2024 Schizont of Plasmodium vivax Schizont stage Almost fills the RBC. Chromatin: numerous irregular masses. Scattered pigment dots. Malaria pigments Schizont of Plasmodium vivax 10/6/2024 Gametocyte of Plasmodium Vivax Gametocyte stage Fill RBC. Rounded with compact cytoplasm and one Schuffner’s dots chromatin mass. Gametocyte of Plasmodium Vivax 10/6/2024 Plasmodium falciparum Diagnostic stages Ring stage Gametocyte stage Ring form of Plasmodium falciparum 1) Thin loop of cytoplasm. 2) Single or double chromatin mass with a vacuole. 3) It occupies 1/6 of RBC. 4) Multiple infection of RBC is common. 5) No enlarged RBC. Gametocyte of Plasmodium falciparum Crescent or banana shaped. RBC is stretched. Ring stage Normal size of RBCs P. vivax P. falciparum - Very thin rim of - Thin rim of cytoplasm. cytoplasm. - Single chromatin dot. - Usually two chromatin - 1/3 RBC dots. - 1/6 RBC 10/6/2024 Ring stage P. vivax P. falciparum - Enlarged infected RBC -Infected RBC same size as others. - Single infection per RBC -Multiple infection per RBC 10/6/2024  Identify the parasitic stage and the labeled structures Plasmodium vivax Ring stage Rim of cytoplasm Chromatin dots Identify the parasitic stage and the labeled structures Plasmodium vivax Trophozoite Schüffner’s dots Vacuoles  Identify the parasitic stage Plasmodium vivax Schizont stage  Identify the parasitic stage and the labelled structure Plasmodium vivax Gametocyte stage Malaria pigments Identify the parasitic stage and the labelled structure Plasmodium falciparum Ring stage Chromatin dot  Identify the parasitic stage Plasmodium falciparum Gametocytes  Identify the parasitic stages Plasmodium falciparum Gametocytes and Rings Babesia spp. “Babesiosis”  Distribution: Babesia is a blood parasite of many mammals as cattle, sheep and dogs. Man is accidentally infected with Babesia microti, transmitted from rodents, and Babesia divergens, transmitted from cattle. They are found in many parts of the world (USA, Europe) Habitat: RBCs (intraerythrocytic) DH and Vector: Hard Tick IH: Man RH: Rodents and Cattle Morphology: - Sporozoites occurs in salivary glands of ticks, they are pear shaped. - Intraerythrocytic stages: ring form with very thin cytoplasm and large chromatin, and merozoites (2-5 µm), pyriform spherical, or ovoid. May be single or pairs or tetrads (Maltese Cross) Life Cycle Infective stage:Sporozoites (bite of hard ticks), merozoites (blood transfusion) Mode of infection: Bite of infected hard tick (transmit infection from animals as rodents and cattle to man), blood transfusion, rarely congenitally. Mode of transmission: Cyclopropagtaive + Transovarian (Transstadial transmission) Ring form Large single Maltese chromatin and thin rim Cross of cytoplasm Tetrad of merozoites Merozoites Pyriform or ovoid 2-5 µm arranged in pairs or tetrads Babesia Ring form and Merozoites Babesia Ring form and Merozoites Babesia may be misdiagnosed as P. falciparum Babesia spp. Plasmodium falciparum.  Hard Tick (vector of Babesia spp.) Capitulum Scutum Large arthropod, 10-15 mm, composed of 2 regions: The anterior region carries the mouth parts called the capitulum covered by teeth arranged in several rows. The posterior region Hard tick female Hard tick male carries 4 pairs of legs that end in claws. The Scutum partially Scutum completely thorax and abdomen are not clearly covers body covers body differentiated. Life Cycle Ticks parasitize animals e.g. dogs, cats, rodents, birds, cattle, horses and wild animals. Humans are accidentally parasitized. Both sexes of the adult ticks, larvae, and nymphs feed on blood. They live for several years in absence of blood. Adult, larva and nymph feed on different host. They are permanent ectoparasites. Female lay egg under stones. Ticks are efficient vector (WHY?) Incomplete metamorphosis (Egg----Larva----Nymph---Adult) Hard Tick (vector of Babesia spp.) Ticks feed by cutting the host epidermis with their teeth and inserting the hypostome into the cut, thereby attaching to the host Hard Tick (vector of Babesia spp.) TRYPANOSOMES African trypanosomes (Polymorphic) 1. Trypanosoma brucei gambiense The causative organism of West African Sleeping Sickness (chronic disease). 2. Trypanosoma brucei rhodesiense The causative organism of East African Sleeping Sickness (acute disease). American trypanosomes (Monomorphic) Trypanosoma cruzi The causative organism of Chagas’ disease  TRYPANOSOMES Polymorphic Trypanosomes African Trypanosomes Trypanosoma b. gambiense Trypanosoma b. rhodesiense “Human African Trypanosomiasis (HAT)” 1. Amastigote (Leishmania form) 2-5 x 1-2 µ, oval or spherical. No free flagellum. 2. Promastigote (Leptomonad form) 10-20 x 1-3 µ, elongated. Nucleus is central. Kinetoplast anterior. Anterior free flagellum. Epimastigote (Crithedial from) 10-20 x 1-3 µ, elongated. Kinetoplast anterior to the central nucleus (juxta-nuclear). Flagellum raises short undulating membrane before being free Trypomastigote (Trypanosome from) 15-35 x 1-3 µ, spindle-shaped. Kinetoplast posterior. Long undulating membrane. Free flagellum. Morphology of African Trypanosomes Morphology of African Trypanosomes 32 µm, elongated 13 µm, club-shaped, with well no protruding developed flagellum, rounded flagellum and post. End with pointed post. end terminal kinetosome Life Cycle Infective stage: Short metacyclic form Mode of infection: Bite of infected Glossina (Anterior station development in foregut= infective stage pass with saliva, salivarian trypanosomes). Rarely congenital and blood transfusion Mode of transmission: Cyclopropagative, 2-3 Wks. Chancre Winterbottom’s Sign Sleeping Sickness Important to match clinical presentations with causative parasite in OSPE  Mott cells (Plasma cell secrete IgM in CSF Polymorphic Trypomastigotes Lumbar Puncture and CSF examination: trypomastigotes, increased lymphocytes (> 20 in peripheral blood film cells/µl), increased protein (>35 mg/dl), increased IgM. Presence of Morula or Mott cells is pathognomonic for HAT. Lumbar puncture is important to determine the disease stage (early, CNS) and design of treatment. Glossina spp. (Tsetse fly) Large insect 7-11 mm, with head carrying a pair of compound eyes, and a pair of 3-segmented antennae. Mouth part is protruding, labium is toothed. Thorax is three segments, each carries a pair of legs, mesothorax carries a pair of wings with characteristic venation (Cleaver-shaped). The wings cross each other (scissor-like) while at rest. Abdomen is dark, 8 segments. Life Cycle: Complete metamorphosis (Egg----Larva----Pupa--Adult). Bionomics: The flies pass most of their time at rest in shaded places in forested areas. The preferred sites are the lower woody parts of vegetation, many tsetse flies hide in holes in trees’ trunks and between roots. They search for food only for very short periods during the day. Both male and female are blood suckers. G. palpalis (vector of T. gambiense) lay larvae near water collection (riverine) while G. morsitans (vector of T. rhodesiense) lay larvae in forests of the Savannah area. Glossina (Tsetse fly)  TRYPANOSOMES Monomorphic Trypanosomes American Trypanosomes Trypanosoma cruzi “Chagas’ Disease” Trypanosoma cruzi Chagas’ Disease Distribution: Central and South America. Due to migration of people from South America, the disease has been encountered in USA, Europe, Africa, East Mediterranean and Australia Habitat: Macrophage phagocytic system and cells of mesenchymal origin as liver, thyroid, muscle, smooth muscles, bone marrow, lung, brain, etc. DH: Man Vector: Triatomine winged bugs (Triatoma megista, T. infestans) RH: Armadillo, dogs, cats and rodents. Morphology: In man: Trypomastigotes in blood (C-shaped, 20 µm, large kinetosome. Inside cells they change and multiplicate in amastigotes forms. In the midgut of the bug, trypomastigotes change into epimastigotes which pass to hindgut then stool. Morphology of Trypanosoma cruzi 54 Morphology of Trypanosoma cruzi Trypanosoma cruzi in peripheral blood Trypanosoma cruzi in cells ( Monomorphic Trypomastigotes) (amastigotes 2-5 µm oval C-shaped, 20 µm, large kinetosome or rounded)  Trypanosoma cruzi Monomorphic Trypanosomes 3. Monomorphic trypanosomes Trypanosoma cruzi amastigote form in heart muscle Life Cycle Infective stage: Short metacyclic form Mode of infection: 1. Contamination of bites’ wound by feces or urine of infected Triatoma spp. (Posterior station development in hind gut= infective stage pass with stool, stercorarian trypanosomes). 2. Congenital 3. Blood transfusion 4. Organ transplantation 5. Consumption of food contaminated with bug feces. Mode of transmission: Cyclopropagative, 10 days. Chagoma Chagoma Mega Colon Romana’s Sign Important to match clinical presentations with causative parasite in OSPE  Xenodiagnosis: Non infected laboratory Monomorphic Trypomastigotes in bred winged bugs are allowed to feed on Megaoesophagus peripheral blood film suspected patients, then hindgut is Barium Meal dissected 2-3 Wks. to detect the parasite  Triatomine bugs (Winged bugs) (Kissing bugs, Assassin bugs, Cone-nosed bugs Distribution: Central and South America Morphology: Large insect measuring 2-3 cm, dark brown or black in color with reddish markings on thorax and abdomen. Head: elongated like a cone (Cone-nosed bug). Thorax: carries 2 pairs of wings (Winged bug); the 1st is leathery at its base, membranous at its tip, while the second pair is membranous. Abdomen: has sharp edges, alternating dark and pale areas and covered with wings in a scissor like manner at resting position. Life Cycle: Incomplete metamorphosis (Egg-------Nymph---Adult) Bionomics: Live burrows and nests of wild animals, including birds, bats, squirrels and armadillos, on which they feed during the night by sucking Males, females and nymphs suck blood at night while the host is sleeping, usually on the exposed face near the nose, eyes or mouth (Kissing bug). The bite is painless and lengthy lasting for about 10-30 minutes. The bugs defecate while feeding or soon after. Pathogens in the feces get rubbed into the bite wound. The bug can “assassinate” or kill other insects (Assassin bug). Triatoma spp (Winged bug) Triatoma megista Winged bug During rest the abdomen is covered by the wings in a scissor like manner Abdomen has alternating dark and pale areas Leishmania donovani complex “Visceral leishmaniasis”  Leishmania donovani complex (Visceral Leishmaniasis) Distribution: India , Mediterranean Coast, China Central Africa, South America. Disease: Visceral leishmaniasis, kala-azar Habitat: Macrophage-phagocyte system in all viscera (blood, spleen, liver, bone marrow, lymph node, skin, etc.). DH: Man. RH: Dogs and rodents in certain areas. Vector: Female sand fly of the genus Phlebotomus (in Old world) and genus Lutzomyia (in New world). 1. Amastigote (Leishmania form) 2-5 x 1-2 µ, oval or spherical. No free flagellum. 2. Promastigote (Leptomonad form) 10-20 x 1-3 µ, elongated. Nucleus is central. Kinetoplast anterior. Anterior free flagellum. Epimastigote (Crithedial from) 10-20 x 1-3 µ, elongated. Kinetoplast anterior to the central nucleus (juxta-nuclear). Flagellum raises short undulating membrane before being free Trypomastigote (Trypanosome from) 15-35 x 1-3 µ, spindle-shaped. Kinetoplast posterior. Long undulating membrane. Free flagellum. Morphologic forms of Leishmania First term lab. 4 68 Leishmania species amastigote 1 1- Nucleus 2- Kinetoplast 2 2 1 Flagellum Nucleus Kinetoplast Promastigote form of Leishmania in culture Sandfly (Phlebotomus and Lutzomyia spp.) Distribution: worldwide especially in tropical and subtropical counties. In Egypt, Phlebotomus papatasi is the most prevalent spp. Morphology: Small delicate insects measuring 2-3 mm in length, hairy with long legs and antennae, humpy thorax, lanceolate wing and sandy color. Claspers, male genital, and cerci, female genital organs, at the posterior end. Life Cycle: Complete metamorphosis (Egg----Larva----Pupa----Adult) Bionomics Female is a blood sucker. Male is feed on plants Biting occurs outdoors. Most species are active at dawn and dusk and during the night. Blood is taken from humans and animals such as dogs, farm livestock, and wild rodents. Adults are weak fliers, they tend to hop for short distances, that why Phlebotomus-borne diseases are limited in distribution. Phlebotomus papatasii (Sandfly) Small delicate insects measuring 2-3 mm in length, hairy with long legs and antennae, humpy thorax, and sandy color Phlebotomus papatasii (Sandfly) Humpy thorax lanceolate wings Long slender legs  Female male Sandy in colour. Large eyes. Humpy thorax. Long, slender legs Cerci Long, lanceolate wings with pointed Claspers tips. 74 Nematodes of Lymphatic System Wuchereria bancrofti Brugia malayi “Lymphatic filariasis” Wuchereria bancrofti Distribution: Tropical and subtropical areas e. g. Central and South America, coastal Asia and Africa. In Egypt It is found in the Nile Delta particularly on the eastern side of the Damietta branch, in Qalubiya, Sharkia and Dakahlia Governorates. Focal distribution is present in Giza, Cairo and Assiut. Habitat: Adults inhabit lymphatic tissues especially of lower limbs and external genitalia. Microfilariae are found in lungs by day and in blood by night. DH: Man is the only definitive host. Vector: Female Culex mosquito, rarely female Anopheles and Aedes in certain geographical areas. Life Cycle of Wuchereria bancrofti © 77  Microfilaria of W. bancrofti Size: 250 - 300 X 7 – 10 um. Shape: - Thread-like with graceful curves. - The body is full of nuclei arranged in rows. - The anterior end is rounded and devoid of nuclei. - The posterior end is tapering and devoid of nuclei. - It has a redundant sheath. © 78  Wuchereria bancrofti microfilaria in blood film Thread-like 300 um Body filled with nuclei Sheathed +Redundant Smooth curves Ant. end rounded + devoid of nuclei Post. end pointed + devoid of nuclei Brugia malayi Distribution: India and the Far East Habitat: Adults inhabit lymphatic tissues of lower limbs limbs. DH: Man. RH: Monkey and cats (make control more difficult than W. bancrofti Vector: Mansonia spp. Morphology: Adults are similar to W. bancrofti but smaller Microfilaria is similar to W. bancrofti. The posterior end is pointed with two nuclei, the distal is big extending to tail, the proximal one is smaller. Life cycle: As W. bancrofti Microfilaria of Brugia malayi Microfilaria of B. malayi Mosquitoes 82  Adult: 4-10 mm; body is formed of head, thorax and abdomen Head carries a pair compound eyes, 15 segmented antennae with dense hairs in male (plumose antenna) and few hairs in female (pilose antenna) adults, and 4-segmeted maxillary palps (Mx. P). Thorax is formed of 3 segments; prothorax, mesothorax and metathorax. Each segment carries a pair of legs. Mesothorax carries a pair of wings. Abdomen is 10 segments, the last 2 are modified into male (claspers) and female (cerci) Adult Male or Female Dense hair on antenna (plumose hair) Scanty hair on antenna Male (pilose hair) Female  Maxillary palps long with no clubbing ends Antenna with short & sporadic hair Short maxillary Antenna with palps long & dense hair Mouth parts Female Culex Mouth parts Male Culex Non spotted wing Culex Culex egg raft Eggs are dark brown Eggs are laid in batches Culex larva Formed of head, thorax & abdomen Has respiratory siphon long and narrow Culex larva Culex larvae suspend diagonally from the surface of water with siphon up Culex Pupa Non feeding Respiratory trumpets are tubular  Long maxillary palps Maxillary palps long with clubbing ends Antenna with Antenna with short & sporadic hair long & dense hair Mouth parts Female Mouth parts Male Anopheles Anopheles Spotted wing Anopheles Anopheles egg Eggs are dark brown laid singly with air cells Anopheles Larva No Siphon Anophles larva Anopheles larvae Parallel to surface of water Anopheles Pupa Non feeding Respiratory funnel- shaped trumpets  The antennae: Plumose in males Pilose in females The maxillary palps A pair of 4-segmented structure Short or long Thin or club-shaped 100 Male or Female Dense hair on antenna (plumose hair) Scanty hair on antenna Male (pilose hair) Female 101  MP of female mosquitoes Pilose antennae (short hairs) Long Short maxillary palp maxillary palp Anopheles Culex  Long maxillary palps Antenna with short & sporadic hair Short maxillary Antenna with palps short & sporadic hair Female Culex mouth parts Female Anopheles mouth parts  MP of male mosquitoes = Plumose antennae (Long hairs) + Long maxillary palps Club end Thin end maxillary palp maxillary palp Anopheles Culex  Maxillary palps Maxillary palps long with no long with clubbing ends clubbing ends Antenna with Antenna with long & dense hair long & dense hair Male Anopheles mouth parts Male Culex mouth parts  Larvae Long Short & and thick No thin siphon Siphon Siphon Anopheles larva Culex larva Anopheles larva Aedes larva  Wings Short & thick siphon Anopheles larva Culex wing Anopheles wing Aedes larva Toxoplasma gondii “Toxoplasmosis” Toxoplasma gondii Distribution: Worldwide DH: Cats and other felines harbor asexual and sexual stages. (asexual and sexual cycles in intestine, asexual cycle in tissues) IH: Rodents (asexual cycle in tissues) Accidental (nonspecific host): All mammals including man, domestic and farm animals, birds and reptiles (asexual cycle in tissues). Habitat: obligate intracellular parasite, it can invade any nucleated cell. Morphology 1. Trophozoites: 4-6µ x 2-3 µ, crescent or ovoid in shape with central nucleus. 2. Pseudocysts: when the trophozoites invade any cell, they multiply rapidly (tachyzoites) inside it giving a cell containing up to 100 tachyzoites know as pseudocyst. It has not a true cyst wall 3. Tissue cysts: has a true cyst wall, 10-200 µ containing up to 60000 bradyzoites, It might be present in any tissue but mostly present in muscle and nervous tissue. They are formed 1-2 weeks after infection with development of immune response and may persist for life. 4. Oocyst: 9 x12 µ, shed with the stool of cats, It is immature, non- infective when freshly passed. It needs 1-5 days for sporulation to give the infective sporulated oocyst (disporocystic-tetrazoic oocyst )  Life Cycle Extraintestinal cycle in IH: asexual reproduction by endodyogony and Intestinal cycle in cat: pologony which Schizogony followed end in formation by gametogony and of pseudocysts formation of zygote and true cyst after and oocyst that pass development of with stool of cat immunity  Toxoplasma gondii tachyzoites 4x2µ Crescent shape Anterior pointed end Rounded posterior end Central nucleus 112  Bradyzoites tachyzoites Pseudocyst Tissue Cyst  Toxoplasma gondii tissue cyst Rounded Well defined cyst wall Thousands of bradyzoites in brain tissue © 114  Toxoplasma gondii brain cyst Bradyzoites 115 Summary Wuchereria bancrofti microfilaria ©  flagella Nucleus Promastigote form of Leishmania species 118  Nucleus Kinetoplast Amastigote form of Leishmania species Leishmania donovani promastigote- Nucleus Toxoplasma gondii tachyzoites Toxoplasma gondii cyst in brain tissue/ bradyzoites Sand fly male/ claspers Identify the parasitic stage and the labeled structures Polymorphic trypanosome Flagellum Undulating membrane Identify the parasitic stage and the labeled structures Monomorphic trypanosomes/ Kinetoplast 125 Identify the parasitic stage Trypanosoma cruzi amastigote in heart muscle © Which figure represents the diagnostic stage for African trypanosomiasis? A B C © Glossina spp. Cleaver shape cell Triatoma megista Winged bug THANK YOU