“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

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

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

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

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

Advantages:

-Improved management of non-malarial disease

-Savings in drug costs

-Improved adherence to therapy

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

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

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

1. HRP2

2. pLDH

3. aldolase

P. falciparum HRP-2

pLDH

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

Malaria Review

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

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

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)

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

Malaria review

• Signs/symptoms– Splenomegaly– Periodic shaking chills (rupture of RBCs)

followed by spiking fevers (merozoites penetrating other RBCs)

– Sweats– Anorexia– Joint pain

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

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

P. falciparum

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)

P. vivax and P. ovale

• Schuffner’s dots may be present

• Gametocytes are amoeboid shaped, not banana

• Schizonts have 12-14 merozoites

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

P. malariae

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.

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.