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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/