Malaria

Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. It is one of the world’s leading causes of death, particularly among children in developing countries.
Introduction Malaria
Chronology in Malaria
Epidemology
Life cycle
Pathogenesis and clinical feature
Lab Test
Treatment of Malaria
Prevention of Malaria

1. Malaria Technical class 24/2/23

2. Content Introduction Malaria Chronology in Malaria Epidemology Life cycle Pathogenesis and clinical feature Lab Test Treatment of Malaria Prevention of Malaria

3. Introduction Malaria Kingdom: Protozoa Phylum: Apicomplexa ( sporozoa ) Class: C occidea Order: Haemospororida Family: Plasmodiidae Genus: Plasmodium Species: P.  malariae Malaria is a life-threatening disease caused by parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. It is one of the world’s leading causes of death, particularly among children in developing countries. Blood parasites of the genus  Plasmodium . Four species are considered true parasites of humans Humans acts as a natural intermediate host:  P. falciparum, P. vivax, P. ovale  and  P. malariae . P. knowlesi is newer spp .  

4. Chronology in Malaria Millennia ago 18 th century 1880 1885 1891 1898 1902 1984 1955 1976 1992 Charaka and Susruta malaria a/w mosquitoes Louis Alphonse Laveran identify malarial parasite Romanowsky developed staining method ME Ronald Ross demonstrate Anopheles as vector Italians named mal- aria Camillo Golgi describe blood stage Amigo and Grassi described the life cycle Shortt and Garnham exo -erythrocytic stage in human Malaria eradication program Trager and Jensen cultured invitro Failure of program RTS,S developed by GSK

5. Epidemiological classification of malaria depending on endemicity Epidemiological classification of malaria depending on transmission Epidemiology

6. Epidemiology P. falciparum is the most common species in India The major endemic areas in India are in the north­eastern states, Andhra Pradesh, Chhattisgarh, Gujarat, Jharkhand, Madhya Pradesh, Maharashtra, Rajasthan and Odisha 22% of people reside in high transmission area (≥ 1 case per 1,000 population), 67% in low transmission area (0–1 case per 1,000 population) and 11% in malaria free area Most affected>> Odisha (24%)>>92% of cases were due to P. falciparum infection P. malariae (< 1%) >> restricted to Tumkur and Hassan districts of Karnataka P. ovale >>few cases from Odisha, Delhi, Assam, Gujarat and Kolkata

7. Life cycle Causative agent: Anopheline culicifacies in rural areas, A. stephensi in urban areas A. fluviatilis in hilly areas. Others are A. minimus , A. philippinensis , A. sundaicus and A. maculatus Definitive host: Mosquitoes Intermediate host: Human

8. Pathogenesis and clinical feature Benign malaria normocytic normochromic anaemia. factors attribute to the development of anaemia Parasite induced RBC destruction Lysis of RBC due to release of merozoites Splenic removal of both infected RBC and uninfected RBC coated with immune complexes Bone marrow suppression Increased fragility of RBCs Autoimmune lysis of coated RBC Splenomegaly massive proliferation of macrophages that engulf parasitized and non parasitized coated RBCs.

9. Pathogenesis of falciparum malaria Sequestration of the parasites Cytoadherence Rosetting Deformability High level of parasitaemia: 30–40% of total RBC are infected Other virulence factors like: Knob associated histidine rich protein II (HRP-II) Glycosyl phosphatidyl inositol (GPI):stimulates the host immune system to release cytokines like IL-1, TNF and IFN- γ PfEMP-1 Microvascular inflammation Obstruction Perivascular leak

10. Complications of Falciparum malaria Cerebral malaria Pernicious malaria: blackwater fever, algid malaria and septicemic malaria Black water fever: occurs following quinine treatment>> sudden intravascular hemolysis followed by fever, hemoglobinuria and dark urine Algid malaria: Characterized by cold clammy skin, hypotension, peripheral circulatory failure and profound shock Septicemic malaria: Characterized by high degree of prostration, high grade fever with dissemination of the parasite to various organs leading to multi organ failure Pulmonary edema and adult respiratory distress syndrome Hypoglycemia Renal failure: sequestration >acute tubular necrosis. Bleeding/disseminated intravascular coagulation Severe jaundice: results from hemolysis, hepatocyte injury and cholestasis Severe normochromic, normocytic anemia: hematocrit <15% or hemoglobin < 5 g/dL with parasitemia level of more than 100,000/µL (> 2%) Acidosis: Results from accumulation of organic acids like lactic acid.

11. Laboratory Diagnosis Microscopic tests: Peripheral blood smear—Gold standard Fluorescence microscopy (Kawamoto’s technique) Quantitative buffy coat examination Non microscopic tests: Antigen detection tests (RDTs) or ICTs— detects parasitic LDH, HRP-II, aldolase Antibody detection—ELISA Culture—RPMI 640 medium Molecular diagnosis—PCR using PBRK1 primer

12. Peripheral Blood Smear Specimen : Peripheral blood >>ear lobe or by finger prick in older children & adults and from the great toe in infants. Blood films should be prepared directly from the capillary blood. ethylene diamine tetra acetic acid (EDTA) anticoagulated blood, smears should be made within an hour of collection of blood. pregnant women, cord blood and placental impression smears postmortem cases, smears from cerebral grey matter Time for taking blood: few hours after the height of the paroxysm of fever and before taking antimalarial drugs. Frequency : at least twice daily until parasites are detected.

13. Types of peripheral blood smear For thick smear, a big drop of blood>> half inch square area >>dried>>kept in distilled water in a koplin jar for 5–10 minutes>> dehemoglobinization For thin smear, a small drop of blood>>corner of a slide>>spread by another slide at an angle of 45 ◦ and then is lowered to an angle of 30 ◦ and is pushed gently to the left, till the blood is exhausted Thick smear—more sensitive Thin smear—speciation can be done surface of a good thin film is: Even and uniform Consist of a single layer of RBCs “feathery tail end” is formed near the center of the slide Margins of the film should not extend to the sides of the slide

14. Thin smear Advantages Intra RBC morphology of parasite can be seen Species identification RBC morphology Mixed infection % of parasitized RBC Severity Know response to the treatment Disadvantages Fixation of smear Low parasitaemia Less sensitive

15. Thick smear Advantages More sensitive Rapid detection of parasites No fixation of smear Low parasitaemia can be detected Disadvantages Intra RBC morphology of parasite can not be seen Cannot confirms Plasmodium spp.

16. Romanowsky stain Methanol: fix the cells to slide Methylene blue : stain RNA,DNA - blue grey colour Eosin: stain haemoglobin - orange red Parasite cytoplasm : blue Nuclear material: purple red Pigment: depending on type and species

17. Romanowsky stain Leishman’s Giemsa Field’s JSB base Methanol based Water based Water based Water based pretreatment dehaemoglobinisation in thick smear Methanol fixation in thin film Methanol fixation in thin film Methanol fixation in thin film content Methylene blue Eosin Methanol Giemsa Methanol glycerol Field A Methylene blue Azur Field B Eosin Buffered water Solution I Methyleneblue ,  potassium dichromate , sulfuric acid Solution II Eosin pH Neutral 6.8-7.2 - -

18. Forms seen in peripheral blood smear examination

19. Kawamoto technique

20. Schizonts Character P. Falciparum P. vivax Size of RBC Number of merozoites Arrangement of merozoites Presence in peripheral circularion does not change 16-24 (upto 36) asymmetrical absent Increases to become twice the original size 14-20 (upto 24) symmetrical in form of rosette present

21. Gametocytes Character P. Falciparum P. vivax Size F 10 to 12 µm ◊ 2 to 3 µm F 10 to 12 µm M 8 to 10 µm ◊ 2 to 3 µm M 9 to 10 µm Shape F cresentric M sausage-shaped F & M spherical Nucleus F central, compact, surrounded by malarial pigment F peripheral, small, compact, surrounded by malarial pigment M central, diffuse with malarial pigment dispersed all over the cytoplasm M central, diffuse with malarial pigment dispersed all over the cytoplasm Infected RBC deformed, with its membrane stretched over the gametocyte enlarged

22. Ring forms Character P. Falciparum P. vivax Size 1.25 to 1.5 µ m 2.5 µm Cytoplasm Uniform thickness Thick opposite to nucleus Nucleus >1/ring 1 /ring No of rings >1 ring/RBC 1 Location in RBC Inside as well accole forms Inside RBC

23. Quantification of parasites Thick smear is preferred to thin smear for quantification of parasitemia. Quantification is helpful for: Assessing the severity of infection Monitoring the response to the treatment Detecting drug resistance of P. falciparum By thick smear Number of parasites counted per 100 WBCs x Total WBC count (8000)/100 By thin smear Number of parasites counted per 100 RBCs x Total RBC count /100

24. Fluorescence microscopy Kawamoto technique >>fluorescent staining method>>demonstrating malaria parasites. Blood smears are prepared on a slide and are stained with acridine ­orange and examined under a fluorescence microscope. Nuclear DNA – Green , Cytoplasmic RNA –red

25. Quantitative Buffy Coat Examination advanced microscopic technique for malaria diagnosis. consists of three basic steps concentration of blood by centrifugation, (2) staining with acridine orange stain (3) examination under ultraviolet (UV) light source

26. Quantitative Buffy Coat Examination Crescent-shaped gametocytes (1) will appear near the interface of the lymphocyte/monocyte and platelet layers.  A small number of (2) schizonts and (3) mature trophozoites may appear in the granulocyte layer. Ring-shaped (4) immature trophozoites will appear throughout the red blood cell layer, with a concentration near the interface with the granulocyte layer.

27. Quantitative Buffy Coat Examination Advantages Useful for screening large sample faster (the entire tube can be screened within minutes), more sensitive (at least as good as a thick film), uses more blood (60 µL) than thick smear and quantification is possible Disadvantages expensive, less specific and speciation is difficult Cannot store Interpretation Acridine orange has a property of staining the nuclear DNA fluorescent brilliant green. Normal RBCs don’t take up the stain (as they are anucleated ). However, parasitized RBCs appear as brilliant green dots. WBCs also take up the stain

28. Antigen Detection by Rapid Diagnostic Tests Parasite lactate dehydrogenase ( pLDH ): produced by trophozoites and gametocytes of all Plasmodium species differentiate pan malarial pLDH common to all species and pLDH specific to P. falciparum Parasite aldolase : Produced by all Plasmodium species Plasmodium falciparum specific histidine rich protein-2 ( Pf -HRP-II) : produced by trophozoites and young gametocytes of P. falciparum Most of the kits>>combination of two antigens,P . falciparum specific antigen (i.e. HRP2 or pLDH specific for P. falciparum) and other is a pan malarial antigen (like aldolase or pan malarial pLDH )

29. Antigen Detection by Rapid Diagnostic Tests

30. Advantages Rapid simple to perform, no extra equipment or trained microscopist Sensitivity: > 90% sensitive at >100 parasites/µL, markedly reduced at < 100 parasites/µL pLDH is produced by the viable parasites>>to monitor the response for treatment Disadvantages expensive Cannot differentiate between the non falciparum malaria species HRP2 remains positive even after treatment False positive bands appear in rheumatoid arthritis factor positive cases The lower limit to detect HRP2 is 40 parasites/µL and pLDH is 100 parasites/µL. Antigen Detection by Rapid Diagnostic Tests

31. Culture mainly used for preparation of malaria antigens RPMI 1640 medium(Roswell Park Memorial Institute and 1640 denotes the number of passages) continuous flow system mixed with a thin layer of RBC and an overlay medium consists of human serum maintained with 7% CO2 and 1–5% O2 other media used are Delbecco’s modified Eagle medium (MEM), RPMI 1630, and Medium 199.

32. PCR detect a single P. falciparum in 20 µL of blood using PBRK1 primer 100 times more sensitive than that of thick blood smear Speciation can be done Drug resistance genes can be detected Useful tool for epidemiological study.

33. Website: nvbdcp.gov.in

34. Drug resistance in M alaria P. falciparum malaria Chloroquine resistant Sulfadoxine -pyrimethamine resistance Mefloquine resistance P vivax malaria Sporadic cases of resistance to chloroquine and/or primaquine Longer half­ life of drug Mutation of the parasite for resistance Inadequate and irregular usage of drug Poor compliance Host immunity Chloroquine resistant mutations in the genes encoding the transporter proteins such as PfCRT (P. falciparum chloroquine transporter) PfMDR1 (P. falciparum multidrug resistance gene 1). sulfadoxine , pyrimethamine and proguanil resistant point mutation in DHFR

35. Nested PCR was done for detection pfcrt -o and pfmdr-1 for P. falciparum pvcrt -o, pvmdr-1 genes for P. vivax. Gel picture showing band of  pfcrt -o ( Plasmodium falciparum  chloroquine resistant transporter-o) 280 bp and  pvcrt -o ( Plasmodium vivax  chloroquine resistant transporter-o) 120 bp.

36. Sample : 2ml edta blood DNA extraction : DNA Mini Kit (Invitrogen) spin column method Primer Design : : Polymerase Chain Reaction Nested PCR amplifications using a PeqSTAR 96Xx Universal Gradient PCR thermal cycler ( Peqlab , Germany).

37. I n vitro  drug sensitivity testing Antimalarial drug sensitivity testing was performed by invitro micro test (Mark III) performed immediately after the collection of blood The test was considered valid and interpretable if 10% of the parasites in the control well (drug free well) had developed into the schizonts after 24–36 hours incubation. Isolates were considered resistant if they showed schizont maturation at chloroquine concentrations 8 pmol /well (1.6 mmol/L blood). To evaluate the drug-parasite response, the EC50 value (50% inhibition) was calculated by HN Non Lin (V. 1.01 Beta) Software

38. Prophylaxis Against Malaria Chemoprophylaxis vector control strategies vaccine prophylaxis.

39. Chemoprophylaxis travelers going to endemic areas and as a short-term measure for soldiers or police serving in highly endemic areas. Weekly regimen: Chloroquine 300 mg or proguanil 400 mg, or mefloquine 250 mg Chloroquine and mefloquine weekly regimens should be started 1 week and 2 weeks before the travel respectively Daily regimen: Doxycycline 100 mg doxycycline daily regimen should be started 1 day before the travel

40. Vector Control Strategies Anti-adult measures Residual spraying: Spraying the houses with residual insecticides such as dichlorodiphenyl trichloroethane (DDT), malathion and fenitrothion is highly effective against adult mosquito Space application of pesticide in the form of fog or mist by ultra­low volume method of pesticide dispersion Individual protection: Done by reduction of human­ mosquito contact by using insecticide treated bed nets, repellents and protective clothing. Antilarval measures Larvicide: Use of mineral oil or Paris green has been extensively used to kill mosquito larvae and pupae Source reduction (to reduce the mosquito breeding site): Includes environmental sanitation, water management and improvement of the drainage system. Biological larvicide: Gambusia affinis (fish) and Bacillus thuringiensis (bacteria) can be used to kill the mosquito larva

41. Vaccination for malaria Pre-erythrocytic (sporozoites) vaccine prevent the entry of the parasite to liver Useful for people of hypoendemic area Trials are going on using vaccine candidates such as: − PfCSP ( circumsporozoite protein repeats) of Plasmodium falciparum LSA: Liver specific antigen 1, 2, 3 SALSA: Sporozoite and liver stage antigen SSP2: Sporozoite surface protein-2 STARP: Sporozoite threonine and asparagine protein Duffy: Binding protein of P. vivax RTS,S/AS01

42. RTS,S/AS01: repeat and T-cell epitope in the PfCSP ( circumsporozoite protein of Plasmodium falciparum),hepatitis B surface antigen (HBsAg)and a chemical adjuvant (AS01) to boost the immune system response Intramuscular injection; the preferred injection site in children aged ≥ 5 months is the deltoid muscle 4 dose : strategy :three primary doses with a minimum interval of 4 weeks between doses, followed by a booster dose approximately 12–18 months after the third dose. 5-dose strategy: may be considered in areas with high seasonal malaria transmission or areas with perennial malaria transmission with seasonal peaks>> three primary doses administered at monthly intervals and two annual booster doses administered prior to peak malaria transmission season

43. Blood stage vaccine/erythrocytic vaccine help in preventing the disease thus, are useful for people of hyperendemic areas of malaria trials are there using vaccine candidates such as Pf EMP (P. falciparum erythrocytic membrane protein antigen) vaccine Hsp70: Heat shock protein SERA: Serine rich antigen RESA: Ring infected erythrocyte surface antigen ABRA: Acidic basic repeat antigen HRP2: Histidine rich protein-2 Aldolase − Merozoite surface protein (MSP 1,2,3) SPF 66

44. Transmission blocking/anti gametocyte vaccine inducing antigametocyte antibodies in >>passed to the mosquito while they feed>>react with the gametocytic & other sexual stage antigens and interfere with fertilization>>block the transmission of infection>> no direct effect to the individuals. Vaccine candidates used are Pfs230 Pfs45/48

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