Malaria Species • Five Plasmodia species

– P. falciparum

– P. vivax

– P. ovale

– P. malariae

– P. knowlesi

• Majority – P. falciparum (most malaria-associated deaths)

• Tropical Africa, Southeast Asia, Oceania, Haiti, Amazon basin of South America, and Dominican Republic

– P. vivax • Central America, Middle East, and India

– P. knowlesi • South East Asia

Plasmodium life cycle

www.uptodate.com

Sexual cycle in midgut

of female mosquito

Asymptomatic

Symptomatic

(6-16 days)

P. vivax, P. ovale, P. falciparum

- over 48 hr

P. Malariae

- over 72 hr

P. Knowlesi

- 24 hr

Hypnozoites

Malaria life cycle

Diagnostic Tests for Blood Parasites ❑ Blood films (thick and thin); stain with Giemsa.

• Prepare films as quickly as possible after collection from patient (be careful,

some blood parasites are extremely contagious, such as Trypanosomes)

• Heparin leads to morphologic distortion; use EDTA

• Draw blood at 12 h intervals for 36 hours if initial smears negative

• Stain with Giemsa or Wright’s/Giemsa

• Examine thin films all around edges for microfilariae

❑ Immunochromatographic lateral flow EIA for malaria

❑ PCR for malaria, Babesia

Thick and thin smears

Fix with methanol

Do not fix thick smear

DD

D

DD

D

DD

D

DD

D

DD

D

Sequestered

P.

f

P.

v

P.

o

Identification of Plasmodium species ring form

trophozoite schizont

gametocyte

Plasmodium malariae/knowlesi

P. malariae band form trophozoite

P. malariae compact old trophozoite

P. malariae immature schizont

P. malariae young immature schizont

Normal sized RBC

Morphology of

Malarial Parasites

Differentiate knowlesii

vs malariae by

molecular methods

David Bell, Chansuda Wongsrichanalai and John W. Barnwell. Ensuring quality and access for malaria diagnosis: how can it be achieved? Nature Reviews Microbiology. 2006:4;682-695.

Lateral flow immunochromatographic assay

Limitations of Binax

Persistence of HRP-2 after parasite clearance

Humar et al. detected circulating HRP-2 antigen in

68% of treated patients on day 7, and in 27% it was

still present on day 28 (Am J Trop Med Hyg 1997 56:44)

Gene deletion for the production of HRP-2

False-positive results

Cross-reaction with serum rheumatoid factor, causing

Moody Clin Microbiol Rev. 2002 15:66-78

DNA extraction Real-time PCR

Plasmodium species identification by real-time PCR

Qiagen blood mini kit

•Qiagen RotorGene •FastStart SYBR Green Master mix •Singleplex reactions

EDTA Blood

Samples tested (49) n Correctly

identified

Plasmodium genus 52 51 (98.1%)

Plasmodium falciparum 22 21 (95.5%)

Plasmodium ovale 8 8 (100%)

Plasmodium vivax 19 19 (100%)

Plasmodium knowlesi 1 1 (100%)

Plasmodium malariae 6 6 (100%)

•Positive: 56 archived pt samples (SHC and Karolinska Institute)

•Negative: 20 EDTA pt samples from Hematology Lab (4 +ve

1. Pf neg, pan pos

(Seq:Pf)

2. Pf by microscopy,

mixed Pf/Po by

PCR/Seq

1 pan neg and Pf pos, pt

undergoing treatment

(Pf primers more sens)

Accuracy of Plasmodium RT-PCR Reference Standard: Microscopy (n=56)

Lefterova et al JCM 2015 PMID:25972416

Babesia

Blood and Tissue Parasites ❑ Blood

▪ RBCs – Plasmodium; Babesia

▪ WBCs – Leishmania; Toxoplasma

▪ Plasma – Microfilaria; Trypanosoma

❑ Bone marrow – Leishmania; Trypanosoma cruzi;

Plasmodium

❑ CNS – Taenia; Echinococcus; Naegleria;

Acanthomoeba; Balamuthia; Toxoplasma;

Trypanosoma; microsporidia

❑ Eyes – Acanthamoeba; Toxoplasma; Loa loa;

microsporidia

Blood and Tissue Parasites

❑ Lungs – Paragonimus; Cryptosporidium; Echinococcus;

Toxoplasma; Helminth larvae (Ex: Strongyloides)

❑ Muscle – Taenia solium; Trichinella; Onchocerca; T. cruzi;

microsporidia

❑ Skin and skin ulcers – Leishmania; Acanthamoeba;

Onchocerca; microsporidia; Dracunculus

❑ Urogenital – Trichomonas; Schistosoma haemotobium;

microfilaria; microsporidia

Final Quiz – Which parasite is this?

http://www.cdc.gov/malaria/