Article: Detection and identification of Plasmodium w RT-PCR Flashcards
Why diagnose malaria? /Background
-One of the leading infectious diseases in world: 300-500 mil infected, 1-3 mil dead: very important disease
How to diagnose Malaria?
Normally: microscopy in blood slides
-Recently: Ag detection methods are introduced, but mostly for P.falciparum
because its the deadly one
For the rest: Few Ag tests available, and their sensitivity/specificity is low + not quantifiable either
-PCR tests: sensitive, can be also quantitative if you add florescent tags (RT-PCR)
They offer: RT-Q- Nucleic Acid Sequence Based Amplification (RT-Q-NASBA)
Why use RT-Q-NASBA?
1) Much faster than PCR (1 hr instead of 4 hrs)
2) rRNA is more than the DNA of the parasite = more sensitive
3) You don’t need to separately denaturate your DNA, NASBA already has a step that increases temperature to 41 degrees = so no DNA contamination either way
4) RT-Q-NASBA is already done for P.falciparum: its very sensitive
Materials/methods + patient info
P.vivax, P. malariae, P. ovale RT-Q-NASBA (no Knowlesi because human transmission was not discovered yet)
Primer selection: You amplify the rRNA w/ PCR: then clone it on a plasmid: then in vitro transcription/try the primers there for each species
RT-Q-NASBA performed according to kit information on a RT Analyser
Samples were obtained from 79 ppl who were diagnosed with Plasmodium with microscopy, microscopy = golden standard
-Relation calculated with Kappa values, above 0.6 = means great agreement between microscopy and RT-Nasba
Results
P.falciparum: great
P. vivax: showed 1 spillover out of 37: P. ovale in 1 patient is observed
P.malariae: 7 patients w p.malariae confirmed with microscopy
NASBA: found 2 of them negative, 3 of them was identified as P.falciparum mixed infection (which was not)
Mixed infection samples (P.falciparum/P.malariae): 17/20 was identified correctly
2 showed falciparum/vivax instead, 1 only showed P.falciparum
So: P.malariae/P.falciparum distinction often fails
Kappa value was 0.9= amazing agreement btw NASBA and microscopy
