II MBBS Dr Ekta Chourasia Microbiology PLASMODIUM II MBBS Dr Ekta Chourasia Microbiology

Dr Ekta Chourasia, Microbiology Taxonomy Phylum Apicomplexa Subphylum Sporozoa Genus Plasmodium Disease Malaria Geographical Tropical & distribution Subtropical countries 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Genus Plasmodium Consists of 4 species: P. vivax P. falciparum P. malariae P. ovale 26/04/07 Dr Ekta Chourasia, Microbiology

Landmarks in the evolution of Malaria 1880 – Laveran identified the malarial parasite in an unstained smear 1885 – Golgi described the blood stage (erythrocytic schizogony) of malarial parasite – Golgi cycle 1898 – Amigo & Grassi described the life cycle 1891 – Romanowsky introduced the staining method 1897 – Ronald Ross while in Calcutta, India, demonstrated Anopheles sp. of mosquitoes as vectors of malaria. Got Nobel prize for his work in 1902 26/04/07 Dr Ekta Chourasia, Microbiology

Transmission & Life Cycle Definitive host Female Anopheles mosquito Intermediate host Man Infective form Sporozoites Portal of entry Skin Mode of transmission Bite of an infected mosquito Site of localization First in liver cells & then in RBCs 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Phases of Development in Man Inside the liver (tissue phase) Pre- erythrocytic schizogony – no clinical symptoms, no pathological damage Exo- erythrocytic schizogony – cause of relapse Inside the RBCs (erythrocytic phase) Erythrocytic schizogony – cause of malarial paroxsyms Gametogony – infects mosquito 26/04/07 Dr Ekta Chourasia, Microbiology

Morphological forms seen in Humans In liver: Sporozoites Pre erythrocytic schizonts Merozoites – infect RBCs In RBCs : Trophozoites – ring form Schizonts Merozoites – released by the rupture of schizonts – infect other RBCs Gametocytes – micro and macro gametocytes 26/04/07 Dr Ekta Chourasia, Microbiology

Morphological forms seen in Mosquito Further differentiation & development of gametocytes take place in mosquito Macro gametes (female gametes) – each macro gametocyte develops in to one macro gamete in the mid gut of mosquito Micro gametes (male gametes) – one micro gametocyte produces 6 to 8 micro gametes by exflagellation. Zygote – Ookinete – Oocyst – rupture – release of Sporozoites – predilection to salivary glands. 26/04/07 Dr Ekta Chourasia, Microbiology

Other modes of transmission Sporozoite- induced- malaria : injection of an emulsion of salivary glands of mosquito containing sporozoites Trophozoite- induced- malaria : injection of blood from a malarial patient containing the asexual forms of erythrocytic schizogony e.g. Transfusion malaria – when persons with latent infection are used as donors Congenital malaria – transmission through some placental defects (a healthy placenta acts as a physiological barrier) Drug addicts – by using same syringe 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Incubation period P. vivax P. ovale 10 to 14 days P. falciparum P. malariae 18 days to 6 weeks 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Pathogenicity Infection causes intermittent fever – Malaria Each of the 4 species causes a characteristic fever: P. vivax Benign tertian/ vivax malaria P. falciparum Malignant tertian/ falciparum malaria, black water fever P. malariae Quartan malaria P. ovale Tertian/ Ovale malaria 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Clinical Features Series of febrile paroxysms – fever is caused by the release of merozoites & toxins from ruptured erythrocytic schizont which in turn causes the release of cytokines. Quartan malaria – every 72 hrs Tertian malaria - every 48 hrs * each paroxysm has 3 stages - cold stage (rigors), hot stage (high temp., body & joint pains, vomiting & diarrhoea) and perspiration stage (fall in temp.) 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Clinical Features Anaemia – due to breakdown of RBCs, particularly occurs in falciparum malaria Splenomegaly – all forms 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Falciparum Malaria Most widespread Accounts for 80% of malaria cases worldwide Most pathogenic of human malaria species Untreated infections - severe disease & even death, particularly in young children, pregnant woman & non immune adults. 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Falciparum malaria Severe falciparum malaria is associated with Pernicious malaria /cerebral malaria Blackwater fever Anaemia Hypoglycaemia Hypotension Complications in pregnancy 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Pernicious Malaria Def: refers to a series of phenomenon occurring during infection with P. falciparum which, if not effectively treated, threatens the life of the patient with in 1 to 3 days In children & non immune adults, can cause coma & death – Cerebral malaria. Occurs as a result of capillary blockage. 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Black Water Fever Occurs in previously infected subjects Can also occur in non immune adults with severe falciparum malaria, and also as a complication of quinine therapy. A rare but acute condition characterised by sudden & massive hemolysis of parasitised & non parasitised RBCs followed by fever and haemoglobinuria. Often fatal due to renal failure 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Black Water Fever Difficult to find the parasites in the blood following a hemolytic attack. Urine appears dark red to brown black due to the presence of free Hb. Clinical features – fever, rigor, aching pains in the loin, icterus, bilious vomiting, circulatory collapse, haemoglobinuria & acute renal failure. Treatment – Chloroquine, blood transfusion, peritoneal dialysis in ARF. 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Anaemia Can be severe & occur rapidly, particularly in young children Occurs due to destruction of parasitised RBCs – phagocytosis & destruction in the spleen Decreased production of RBCs in the bone marrow. 26/04/07 Dr Ekta Chourasia, Microbiology

Falciparum malaria in Pregnancy Can result in: Severe anemia Low birth weight babies Greatest risk in 1st pregnancy 26/04/07 Dr Ekta Chourasia, Microbiology

Malaria caused by P.vivax, P.ovale & P.malariae Rarely life threatening Relapses/ recurrences are a feature Recurrences in Malaria May result from – reinfection or - due to certain events related to the parasite’s life cycle 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Recurrence of Malaria Two types of recurrences known in malaria: Recrudescence – seen in P. falciparum & P. malariae due to persistence of blood infection (some erythrocytic forms evade host immunity) even after clinical illness has subsided. The numbers may increase later, leading to reappearance of clinical symptoms Occur mostly up to one year or so but in P. malariae, it can occur even after decades 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Recurrence of Malaria Relapse Occurs due to a special form of parasites – hypnozoites. Hypnozoites are the sporozoites that remain dormant after infecting liver Activated from time to time to initiate pre erythrocytic schizogony - Exoerythrocytic schizogony 26/04/07 Dr Ekta Chourasia, Microbiology

Genetic factors protecting against Malaria Sickle cell anaemia – sickle celled RBCs are removed by the spleen before the development of schizonts Ovalocytosis – RBCs are rigid and they resist parasitic invasion Duffy blood group negative individuals – duffy blood group Ag is the receptor for the attachment of merozoites of P.vivax 26/04/07 Dr Ekta Chourasia, Microbiology

Genetic factors protecting against Malaria Newborn infants – natural protection for 1st few months of life due to high conc. of HbF in their RBCs. Beta thalassaemia – protects against severe falciparum infection 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Laboratory Diagnosis Microscopy – detecting & identifying malarial parasites in peripheral blood films. Concentrating parasites in venous blood by centrifugation when they can not be found in blood films Using a rapid malaria Ag or enzyme detection test Other tests – Hb, PCV, Blood glucose, total WBC & platelet count. 26/04/07 Dr Ekta Chourasia, Microbiology

Examination of Blood film Collection of blood - best prepared directly from capillary blood - in EDTA bulb (used within 30 mins) Time of collection - as soon as possible if malaria is suspected - before administering antimalarials - during pyrexial phase 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Types of Blood film Two types: Thick films : - 30 to 40 times more sensitive than thin films - more suitable for detection of malarial parasite when they are few in number - blood is not fixed, RBCs are lysed during staining (only parasitic forms will be seen) 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Types of Blood film 2. Thin films : - to confirm the Plasmodium species - assists in the identification of mixed infections - blood is fixed, parasites are seen within the RBCs - also helps in assessing the response to treatment especially in areas where drug resistance is suspected (by counting the number of parasitised RBCs before & after the treatment) 26/04/07 Dr Ekta Chourasia, Microbiology

Making of Thin & Thick films 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Fixation & Staining Fixation – thin films are fixed with absolute alcohol for 1 to 2 mins. Staining – films are stained with Romanowsky stain: giemsa, field’s, wright’s Giemsa – 10% solution for 10 mins 26/04/07 Dr Ekta Chourasia, Microbiology

Reporting of Blood film Look for the different morphological forms of parasite in blood smear: Trophozoites / ring forms Schizont Gametocytes Identify species – differences in the characteristics of morphological forms in different species 26/04/07 Dr Ekta Chourasia, Microbiology

Trophozoites / Ring forms Character P. vivax P. falciparum Size 2.5µ (1/3rd of RBC) 1.25 to 1.5 µ Cytoplasm Thick opposite to nucleus Uniform thickness Nucleus One/ ring Can have >1 Number of rings One ring/ RBC >1/ RBC Location in RBCs Always inside RBCs Inside as well as on the surface (accole’ forms) Type of RBC infected Preferentially young RBCs & reticulocytes All types 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Thin Blood Film Thick Blood Film Ring Forms / Trophozoites 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology Schizont Character P. vivax P. falciparum Size of RBC Increases to twice its size Does not change No of merozoites 16 8 to 32 Arrangement of merozoites Symmetric in form of rosette Asymmetrical Presence in peripheral blood Present Absent 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology P. vivax P.falciparum 26/04/07 Dr Ekta Chourasia, Microbiology

Gametocytes (male & female) Character P. vivax P. falciparum Shape – Male Female Spherical Cresentic Sausage shaped Nucleus- M F Central, diffuse Peripheral,small Central,compact Infected RBC Enlarged Deformed, with its membrane stretched. 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology P.vivax P. falciparum 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Counting the % age of parasitised RBCs On thin blood films When falciparum malaria parasitemia is high Method of counting: Select an area where no of RBCs is roughly 250. Count the no of parasitised RBCs in 4 such fields i.e. approximately 1000 RBCs. Divide by 10 to obtain the percentage. *WHO – if it is >5%, then the parasitemia is heavy & prognosis is poor. 26/04/07 Dr Ekta Chourasia, Microbiology

Buffy Coat preparation To concentrate malarial parasite Centrifuge EDTA anticoagulated venous blood in a thin bore capillary tube Buffy coat layer is formed between the RBCs & the plasma. Break the tube & transfer buffy coat & RBCs to a slide - make a thin smear – air dry – fix with ethanol – stain with Giemsa. 26/04/07 Dr Ekta Chourasia, Microbiology

Quantitative Buffy Coat Capillary tube is coated with an anticoagulant & Acridine orange fluorescent dye After centrifugation, the tube can be used for two purpose: Complete blood count Identification of malarial parasite using a fluorescence microscope. 26/04/07 Dr Ekta Chourasia, Microbiology

Quantitative Buffy Coat 26/04/07 Dr Ekta Chourasia, Microbiology

Rapid Diagnostic tests Developed to diagnose falciparum malaria rapidly & without a microscope. Can also detect vivax malaria Three tests are available commercially Detects either HRP2 Ag (Histidine rich protein) or specific pLDH (parasite lactate dehydrogenase) Both HRP2 & pLDH are produced by the parasites during their growth & differentiation in RBCs. 26/04/07 Dr Ekta Chourasia, Microbiology

Rapid Diagnostic tests HRP2 tests detection of P.falciparum Two types of test – ParaSight F - ICT Malaria Pf pLDH test e.g. OptiMAL test Detection of P.falciparum & P.vivax Produced by all human malarial parasites Differentiation of species is based on antigenic differences between pLDH isoforms. 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology ParaSightF test Optimal test ICT Malaria Pf / Pv 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Stage specificity of antimalarial drugs Stage of malarial parasite Antimalarial drug Sporozoite Proguanil, Pyrimethamine Hypnozoite Primaquine Pre- erythrocytic schizont Blood schizont Chloroquine, Quinine, Artemisinin Gametocyte 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology

Dr Ekta Chourasia, Microbiology 26/04/07 Dr Ekta Chourasia, Microbiology