Malaria lab diagnosis

MBBS, BMLT, DMLT and other allied health students

1. Malaria Arun Kumar Parthasarathy D.Y Patil Medical College, Kolhapur

2. Malaria parasites Belongs to the genus Plasmodium Approx 156 named spp- infect vertebrates 4 – infect humans P.falciparum P.vivax P. malariae P.ovale Important vector borne disease. P. Knowlesi -parasite of monkey but can also affect humans and many cases affecting man were recently reported fromAsia . 95% of infection

3. Distribution P. falciparum- T ropics P. vivax- widest distribution (Tropics, Subtropics & Temperate P. malariae - Sporodically P.ovale - Rare, common in western Africa In India- P.vivax & P.falciparum - common Few cases- P.malariae & P.ovale reported

4. Vector – Female Anopheline mosquito Out of 45 species of Anopheline mosquitoes in India, only a few are regarded as vectors of primary importance. A. culicifacies rural areas A. stephensi urban areas A. fluviatilis hilly areas minimus , A. philippinensis , A. sundaicus and A. maculatus Others

5. Life Cycle Complex life cycle Intermediate host: man (Asexual Phase) Definitive host: Mosquito (Female anopheles)- Sexual Phase Infective form : Sporozoites

6. Stages.. Primary exo-erythrocytic stages/ Pre- erythrocytic schizogony Erythrocytic schizogony Gametogony Secondary exo-erythrocytic or dormant schizogony

7. Pre-patent period Ring forms are the first asexual form that can be demonstrated in the peripheral blood. The time interval betweeen the entry of the parasite into man and demonstration of the parasites in the peripheral blood- pre-patent period. It varies b/w the species P.falciparum - 5 days P.ovale-13 days P.malaraie -9 days P.vivax -8 days

8. P. vivax P. falciparum P. malariae P.ovale Yellowish brown, Fine granules Dark brown, one/two solid blocks Bark brown, Coarse granules Dark yellowish brown, coarser than those of P. vivax Malarial Pigment… Plasmodium feeds on haemoglobulin. The undigested product of haemoglobulin metabolism like hematin , excess protein and iron porphyrin combine to form malarial pigment

9. P. vivax P. falciparum P. malariae P.ovale Large, 2.5 μ m in diameter, usually one chromatin dot &occasionally 2 chromatin dot Small, delicate, 1.25-1.5 μ m in diameter with 2 chromatin dots, 2 ring in one RBCs common Accoles form-some parasites lie on RBC membrane Similar to that of P.vivax Similar to that of P.vivax Early Trophozoites/ Ring stage

10. P. vivax P. falciparum P. malariae P.ovale Large, amoeboid, predominant vacuole Medium size , compact & rounded Small, compact, band-shaped, slightly amoeboid, vacuole disappears early Slightly amoeboid Late Trophozoites

11. P. vivax P. falciparum P. malariae P.ovale Large, 9-10 μ m in dm, almost fills an enlarged RBCs Small, 4.5-5 μ m in diameter fills two third s of normal size RBCs which is not enlarged Small, 6.5-7 μ m , almost fills a of normal size RBCs Small, 6.2 μ m , fills about three quarters of slightly enlarged RBCs Schizonts

12. P. vivax P. falciparum P. malariae P.ovale 14-24 14-32 6-12 6-12 Merozoites

13. On 10 th day – oocyst is fully mature and releases the Sporozoites (Body cavity of mosquito ) Through the body fluids – Sporozoites are distributed to various organs of the body except ovaries Sporozoites are special predilection for salivary glands and reach in maximum number ultimately in the salivary ducts .

14. Laboratory Diagnosis Malaria is one of the few parasitic infections considered to be immediately life-threatening and a medical emergency Microscopy: Blood collection: finger prick or ear lobule Thick and Thin smears of the blood are prepared on same or different slides Thick smear – Dehaemoglobulization is done by keeping the slide in distilled water in Koplins Jar in vertical position – 5-10 minutes till the slide becomes white and then air dried Thick and thin smear are stained with Leishman stain Then slide examine under 100x with oil

15. Parasite are abundant in peripheral blood, late in the febrile paroxysm All asexual erythrocytic stages as well as gametocytes can be seen –peripheral blood ( P.vivax , P.malariae , P.ovale ) P.falciparum – ring form and Crescent shape gametocytes Late Trophozoite and Schizonts – internal organs and appear in peripheral blood only in severe or perinecious anemia Malaria pigments may be demonstrated inside the monocytes and PMNL cells Presence of malaria pigment and absence of malaria parasites-P. falciparum infection RBCs –enlarged in vivax infection

16. Thin film is examined first and if parasites are found – no need to examine thick smear If parasite not seen in thin smear in a few minutes the thick smear should be examined Parasites are more along the upper and lower margins of the tail Atleast 200-300 oil immersion field should be examined before the smears are considered negative

17. Fluorescence microscope: stains with Acridine orange - allows quicker screening of films, because parasites are more readily recognized and a low power lens may be used

18. Quantification Quantification is done by calculating the number of parasites counted compared to number of WBCs in the thick smear or the number RBCs counted in the thin smear Quantification by thick smear = No. of parasites counted No. of WBC Counted X 100 Quantification is helpful for 1. Assessing the severity of infection 2. Monitoring the response to the treatment 3. Detecting drug resistance P.falciparum

19. Rapid Test Most frequently they employ a dipstick or test strip bearing monoclonal antibodies directed against the targeted parasite antigens . Within 15 minutes test can be performed Targeted antigens: Histidine -Rich Protein II (HRP-II): water soluble protein produced by trophozoites and young gametocytes of P.falciparum. 2. Parasite Lactate Dehydrogenase (pLDH): produced by asexual and sexual stages of malaria parasites. They can distinguish P.falciparum from the non-P.falciparum spp but cannot distinguished between other 3 spp

20. Dual antigen test Test detect pLDH (Parasite Lactate Dehydrogenase ) produced by trophozoites and gametocytes of all plasmodium spp and PF-HRP-2 antigen. 2 bands are seen, one is genus specific (Plasmodium) and other is species specific ( P.flaciparum ) It is a Rapid 2 site sandwich immunoassay Used for species detection and differentiation of P.vivax and P.falciparum malaria in areas with high rates of malaria infection. Disadvantage:- cannot differentiate between P.vivax , P.ovale and P.malariae

21. Quantitative Buffy Coat Test (QBC) Developed by Becton and Dickenson Inc. It involves staining of centrifuged and compressed of red cell layer with acridine orange Examination under UV light source Other Technique PCR, Serology

22. Culture Trager and Jensen(1976) successfully cultivated and maintained P.falciparum in vitro in human red blood cells Used RPMI 1640 medium in a continuous flow system Human erythrocytes were in a shallow stationery layered covered by a shallow layer of medium Medium was flowed slowly and continuously under an atmosphere with 7% CO2 , 1-5% O2 Now it is widely used for the production for the antigen

23. Treatment Chloroquine – standard treatment for acute malaria for many years Quinine is the most reliable and alternative to Chloroquine Tetracycline and Clindamycin- adjunct to quinine therapy Mefloquine and Halofantrine – active against Chloroquine resistant strains Chlorquine and Quinine – do not eliminate exo -erythrocytic parasites in liver For this, Primaquine (8-aminoquinoline drug) should be used but drug may be precipitate hemolysis in individuals – deficient in the enzyme Glucose 6 Phosphate Dehydrogenase

24. WHO definition and Classification of Drug Resistance Ability of a parasite to multiply or to survive in the presence of concentrations of a drug that normally destroy parasites of the same species or prevent their multiplication. Three levels of resistance[R] are defined by WHO RI: following treatment, Parasitaemia clears but a recrudescence occurs RII: following treatment, there is a reduction but not a clearance of parasitaemia RIII: Following treatment, there is no reduction of parasitaemia Method classifying resistance, based on counting trophozoites in blood films for upto 7 days after treatment and monitoring the patient for any subsequent recrudescence

25. Prophylaxis Spraying Insecticides such as DDT or Malathion Spraying the breeding sites with petroleum oils and Paris Green (Copper Acetoarsenite )- larvicides Using Larvivorous fish ( Gambusia affinis ), Bacterium (Bacillus thuringiensis var. israelensis or serotype H14)- spore forming bacteria produce a crystal of toxic protein( γ - endotoxin )- spores and crystals are ingested by mosquito larva, the mouthparts and gut are paralysed and gut epithelium is destroyed – leading to death Avoiding exposure to mosquito bite

26. Thank You