Sporozoa I

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1. (Malaria parasite and Babesia) By, Bajarangi lal Chaudhary Ph.D. Scholar

2. Chapter Outline Classification Malaria Parasite Babesia

3. CLASSIFICATION

4. The causative Agent of malaria P. vivax causes benign tertian malaria. P. falciparum causes malignant tertian malaria. P. malariae causes benign quartan malaria. P. ovale causes ovale tertian malaria P. knowlesi causes quotidian malaria.

5. Life Cycle Host: Female Anopheles definitive and Man intermediate host. Human Cycle: Pre- erythrocytic Schizogony Erythrocytic schizogony Gametogony

6. Mosquito Cycle Exflagellation : Zygote: Ookinete : Oocyst : Sporozoites : Extrinsic incubation period:

7. Life cycle

8. Pathogenesis and Clinical Feature Benign malaria: It is characterized by a triad of febrile paroxysm, anemia and splenomegaly . Febrile paroxysm: ( 1) cold stage: 15 minutes to 1 hour. The patient feels lassitude, headache, nausea, intense cold, chill and rigor (2) hot stage: 39–41°C (3) sweating stage : Fever comes down with profuse sweating

9. Anemia 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 leading to decrease RBC production. Increased fragility of RBCs. Autoimmune lysis of coated RBCs.

10. Splenomaegaly Splenomegaly is due to massive proliferation of macrophages that engulf parasitized and nonparasitized coated RBCs.

11. Falciparum malaria (malignant Tertian malaria) Sequestration of the parasites: This leads to blockade of vessels, congestion and hypoxia of internal organs. Mediated by: Cytoadherence : P. falciparum erythrocyte membrane protein-1 (PfEMP-1) Rosetting : Deformability Other virulence factors like : (HRP-II) and Glycosyl phosphatidyl inositol (GPI)

12. Complications Falciparum malaria Cerebral malaria: Pernicious malaria: Black water fever: Algid malaria: Septicemic malaria: Pulmonary edema and adult respiratory distress syndrome: Hypoglycemia: Renal failure: Bleeding/disseminated intravascular coagulation Severe jaundice: Severe normochromic , normocytic anemia: Acidosis:

13. Cerebral malaria

14. Chronic Complications of Malaria Tropical splenomegaly syndrome (hyperactive malarial splenomegaly ) Quartan malarial nephropathy Promotes Burkitt’s lymphoma

15. Malaria in Special Situations Transfusion malaria Malaria in pregnancy Malaria in children

16. Transfusion malaria The infective form is trophozoite There is no pre erythrocytic stage of development and no relapse The incubation period is often short Radical chemotherapy with primaquine is unnecessary as there is no relapse

17. Malaria in pregnancy Malaria during pregnancy increases the risk of fetal distress and can result in premature labor low birth weight and still birth

18. Malaria in children complications are relatively common among children like convulsions, coma, hypoglycemia, metabolic acidosis and severe anemia whereas other complications like deep jaundice, acute renal failure, and acute pulmonary edema are unusual in children.

19. Plasmodium knowlesi It is a malaria parasite of monkey but can also rarely affect humans. Anopheles leucosphyrus is the main vector The first human case was documented in 1965

20. Clinical features P. knowlesi produces an acute illness and relatively high parasitemia. Paroxysms of fever occur daily (quotidian malaria) because of short RBC cycle (24 hours)

21. Lab diagnosis The late trophozoites (with band forms), schizonts (8–10 merozoites arranged in a rosette) and round gametocytes of P. knowlesi, are morphologically similar to that of P. malariae . No commercially available rapid diagnostic tests (RDTs). (PCR) assays are available using the primers Pmk8 and Pmkr9.

22. Immunity Against Malaria Innate Immunity: Age of red blood cells: Nature of hemoglobin : Hereditary ovalocytosis : Red blood cells with glucose-6-phosphate dehydrogenase (G6PD) deficiency Duffy negative red blood cells HLA-Bw53 Nutritional status

23. Acquired immunity Humoral immunity: Circulating antibodies (IgA, IgM and IgG) against asexual forms give protection by inhibiting the red cell invasion and sequestration, whereas antibodies against sexual forms help in reducing the transmission of malaria Cellular immunity: Cytokines released from T cells stimulate the macrophages and also the B cells to produce antibodies. stimulate

24. Epidemiology of Malaria Transmitted in 108 countries containing 3 billion people. P. vivax is the predominant species. Children are more prone to infection and complications.

25. Malaria Situation P. falciparum is the most common Odisha was affected the most (24%) where 92% of cases were due to P. falciparum infection. The largest focus of P. malariae in India is reported to be in Tumkur and Hassan districts of Karnataka.

26. Laboratory Diagnosis Microscopic tests: Peripheral blood smear—Gold standard Thick smear—more sensitive Thin smear—speciation can be done Fluorescence microscopy (Kawamoto’s technique) Quantitative buff y 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

27. Thin and thick blood smear microscopic tests: Peripheral blood smear—Gold standard Thick smear—more sensitive Thin smear—speciation can be done

28. Thin blood smear showing ring form of Plasmodium malariae (band form) Thin blood smear showing ring form of Plasmodium ovale

29. Quantification of malaria parasites by thick smear

30. P. Falciparum , thin Blood smear (A) multiple ring form and accole form; (B) double dot (head phone shaped) ring form; (C) gametocyte A B C

31. Thin blood smear showing different forms of Plasmodium vivax (A) ring form; (B) gametocyte; (C) schizont A B C

32. Fluorescence microscopy Kawamoto technique Blood smears are prepared on a slide and are stained with acridine -orange and examined under a fluorescence microscope. Nuclear DNA is stained green.

33. Quantitative Buff y Coat Examination It consists of three basic steps:- concentration of blood by centrifugation staining with acridine orange stain examination under ultravoilet (UV) light source Interpretation: Normal RBCs don’t take up the stain (as they are a nucleated). However, parasitized RBCs appear as brilliant green dots. WBCs also take up the stain

34. Quantitative Buff y Coat Examination (A) QBC capillary tube (B) magnifi ed view of QBC capillary tube after centrifugation A B

35. Antigen Detection by Rapid Diagnostic Tests Parasite lactate dehydrogenase ( pLDH ): produced by trophozoites and gametocytes of all Plasmodium species. Parasite aldolase: Produced by all Plasmodium species Plasmodium falciparum specifi c histidine rich protein-2 (Pf-HRP-II): young (but not mature) gametocytes of P. falciparum Sensitivity: 90% sensitive at >100 parasites/ μL .

36. Quantitative Buff y Coat Examination (C) crescent shaped gametocyte of Plasmodium falciparum (D) ring forms of Plasmodium falciparum seen as fluorescent dots C D

37. (A) Schematic diagram of rapid diagnostic test kit showing negative, non falciparum, pure or mixed infection with Plasmodium falciparum and invalid result of malaria; A

38. Antibody Detection Detection of antibody in serum indicates past malaria infection and is useful for: Epidemiological survey in malaria Screening of blood bank to identify the infected donors.

39. Culture RPMI 1640 medium (Roswell Park Memorial Institute and 1640 denotes the number of passages) in a 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. The other media used are Delbecco’s modified Eagle medium (MEM), RPMI 1630, and Medium 199.

40. Molecular Diagnosis DNA probe: Highly sensitive if the parasite count is low less than 10/ μL . (PBRK1 primer) Speciation can be done Drug resistance genes can be detected Useful tool for epidemiological study.

41. Nonspecific Tests Normochromic and normocytic hemolytic anemia Leucopenia: Raised erythrocyte sedimentation rate (ESR) Raised serum C-reactive protein Prolonged prothrombin and partial thromboplastin time in severe infection Decreased antithrombin III levels in mild infection Metabolic acidosis. Hypoglycemia Hypergammaglobulinemia

42. Treatment

43. Antimalarial Drug Resistance Falciparum malaria: Chloroquine resistant, Sulfadoxine-pyrimethamine resistance, Mefl oquine resistance Vivax malaria: Only sporadic cases of resistance to chloroquine and/or primaquine in some areas have been reported

44. Mechanism of drug resistance Chloroquine resistance in Plasmodium falciparum Occurs due to mutations in the genes encoding the transporter proteins such as PfCRT ( P. falciparum chloroquine transporter) and PfMDR1 (P. falciparum multidrug resistance gene 1). These proteins help in chloroquine influx into the parasitic food vacuoles. Such mutation results in impaired transport of chloroquine . More so, mutation in PfMDR1 gene leads to resistance to other antimalarials like amodiaquine , mefloquine and halofantrine . Resistance to antifolates such as sulfadoxine : point mutation in DHFR ( dihydrofolate reductase ) gene.

45. Who Guideline for Assessing Degree of Resistance In vivo method (2002) degree of resistance is divided into four categories. 1. Early treatment failure (ETF): 2. Late clinical failure (LCF): 3. Late parasitological failure (LPF): 4. Adequate clinical and parasitological response (ACR):

46. Prophylaxis Against Malaria Chemoprophylaxis: Weekly regimen: Chloroquine 300 mg or proguanil 400 mg, or mefloquine 250 mg. Daily regimen: Doxycycline 100 mg Vector Control Strategies: Residual spraying:- dichlorodiphenyl trichloroethane (DDT), malathion and fenitrothion is highly effective against adult mosquito Space application Individual protection: Larvicide : Use of mineral oil or Paris green Source reduction Biological larvicide :

47. Vaccination for malaria The vaccine candidates are poor inducer of cell mediated immune response Antigenic variation in malarial antigens such as PfEMP Different immune mechanisms occur in different stages of malaria life cycle.

48. BABESIA It rarely affects humans causing opportunistic infection. Babesia species are grouped into : Small Babesia species (1–2.5 μm): B. microti, B. gibsoni and B. Rodhaini Large Babesia species (2.5-5 μm): B. divergens and B. bovis .

49. Pathogenesis and Clinical Features Incubation period varies from 1 to 6 weeks. Mild Babesia microti illness: malaise, fatigue, and weakness and fever. Severe Babesia microti illness: Infections by Babesia divergens , Babesia bovis and Babesia duncani :

50. Epidemiology Babesiosis is highly endemic in the North Eastern United States like Nantucket Island and also in South Eastern Massachusetts It is an emerging infectious disease in other countries. Sporadic cases are reported in Europe and other places In India, Babesiosis is not reported yet

51. Laboratory Diagnosis Peripheral blood microscopy :-Detects maltese cross form (ring form in tetrad) Serology antibody: IgM titers of 1:64 or more and IgG titers of 1:1024 or more signify active or recent infection Molecular method: 18S rRNA gene (PCR) Animal inoculation

52. Giemsa stain blood smear showing maltese cross form

53. Treatment Oral atovaquone plus azithromycin for 7–10 days.

54. References Essentials Medical Parasitology by Apurba Sankar Sastry .