1. “Ensuring quality and access for malaria diagnosis: how can it be achieved?” Nat Rev Microbiol. 2006 Sep;4(9 Suppl):S7-20. Amy Storfa 3/23/2007

2. Background Symptoms (fever, rigors, headache) overlap with presentations of other febrile illnesses P. falciparum can often lead to coma and death; other Plasmodium spp. cause acute severe illness but rarely fatal Despite microscopy, most diagnoses with subsequent treatment made based on symptoms Estimates of malaria deaths estimated at ~1-2 million/y –350-500 million cases of malaria occur annually Programs developed for treatment but accurate diagnosis not emphasized

3. Background Some countries maintain microscopy-based diagnosis programs (e.g. India) BUT, microscopy largely unavailable to providers of most patients with tropical febrile illness Requires organized health system, supplies, reagents, good microscopes, maintenance, competence, ability to make blood films Antigen-detecting rapid diagnostic tests (RDTs) first introduced in early 1990s and used sparingly in malaria endemic areas

4. Background More recently, drug resistance is increasing, necessitating switch to artemisinin-based combination therapy (ACT), more $ Shift has led to upsurge in use of RDTs But many areas (particularly sub-Saharan Africa) still rely on symptom-based diagnosis

6. Early diagnosis Differential diagnosis of febrile tropical illness: respiratory tract infxn, typhus, viral illness, meningitis Early detection/treatment likely to occur if microscopy services are offered within 15-20 minutes of residence ACT ~$1.60 per course RDT ~$0.55 to $1.00 each Overall funding for research and development –Diagnosis receives <1%, vs 37% for drug development

8. Advantages: -Improved management of non-malarial disease -Savings in drug costs -Improved adherence to therapy

9. Implications on health care system Preparation and interpretation can be poor Requires adequate level of training, supervision Appropriate instructions developed Requires temperature controlled distribution and storage facilities Need to be stable, simple to use, able to detect clinically significant disease (100 parasites/uL) Need quality assurance and control

11. Bell et al. Nature Reviews Microbiology 4, S7–S20 (September 2006) | doi:10.1038/nrmicro1525

13. 1.HRP2 2.pLDH 3.aldolase

14. P. falciparum HRP-2

15. pLDH

17. Further development/study needed Field trials needed to see how tests perform in field WHO initiative to test accuracy and stability of RDTs Previous studies based on various RDT types in different clinical and epidemiological settings are difficult to compare Difficult to generate conclusive comparisons of RDT performance Timing of treatment and effectiveness of therapy also complicates comparative studies –pLDH is rapidly cleared but HRP2 persists for weeks

18. Malaria Review

19. Malaria review Plasmodium: four species –P. falciparum (~45%) –P. vivax (~45%) –P. ovale (rare, <5%, limited to W. Africa) –P. malariae (<5%) Acquired via anopheles mosquito

20. Malaria review: multiple forms Trophozoites (=ring forms): most numerous form to see in peripheral blood, ring like structure (<1/2 diameter of cell), progressively enlarge and mature to… Schizont: multinuclear structure, appear as intraerythrocytic collection of merozoites (each with its own nucleus) Gametocyte: mononuclear structure occupying >1/2 the red cell, usually amoeboid in shape and nearly fills entire RBC

21. Life cycle Mosquitoes inject sporozoites, divide in liver into schizonts (containing merozoites) Merozoites infect RBC and then become trophozoites Again divide into merozoites –Can infect more RBCs or become gametocytes (ingested by mosquitoes)

22. Malaria review Infects RBCs; causes intermittent hemolysis with paroxysmal fevers Fever q48 h (tertian fever): P. falciparum, P. ovale; P. vivax Fever q72 h (quartan fever): P. malariae Examine thick (for screening) and thin (for species identification) films

23. Malaria review Signs/symptoms –Splenomegaly –Periodic shaking chills (rupture of RBCs) followed by spiking fevers (merozoites penetrating other RBCs) –Sweats –Anorexia –Joint pain

24. P. falciparum Malignant tertian fever because potentially lethal Must be identified Usually only early ring forms and gametocytes seen –Ring forms: may have double chromatin dots, may be multiply infected; accole or applique forms present; less than 1/5 size of RBC –Gametocytes: banana shaped Infected red cells NOT enlarged, infects RBCs of all stages of maturation

25. P. falciparum Acute intravascular hemolysis with hemoglobinuria (“blackwater fever”) Infected RBCs have “sticky knobs” leading to sludging, infarcts of brain, kidneys With no treatment, patients either die or spontaneously resolve within one year

26. P. falciparum

27. P. vivax and P. ovale Benign tertian fever Morphologically very similar P. ovale very rare, confined to Western Africa Both infect young RBCs and appear enlarged and pale All stages seen (early and developing rings, schizonts, gametocytes)

28. P. vivax and P. ovale Schuffner’s dots may be present Gametocytes are amoeboid shaped, not banana Schizonts have 12-14 merozoites

29. P. malariae Associated with nephrotic syndrome Infects older erythrocytes, normal to small sized RBCs No Schuffner’s dots All stages seen Schizonts have 6-12 merozoites, rosette pattern Coarse pigment may be present Occasional band forms (trophozoite form) seen Low grade cryptic infections can occur up to 40 y

30. P. malariae

31. Malaria and RBC associations Hemoglobin S trait (HbSA) protective against P. falciparum Duffy antigens mediate attachment of P. vivax (Duffy negative patients protected from P. vivax) Glucose-6-phosphate dehydrogenase deficiency protects against ALL Plasmodium spp.

32. References Bell D et al. “Ensuring quality and access for malaria diagnosis: how can it be achieved?” Nat Rev Microbiol. 2006 Sep;4(9 Suppl):S7-20. Jones SL. Clinical Laboratory Pearls. Lippincott Williams and Wilkins, 2001. Mais DD. Quick Compendium of Clinical Pathology. ASCP Press, 2005.