Evaluation of saliva-derived Plasmodium falciparum DNA
using the OMNIgene®•ORAL kit in detection of malaria
Wilson Okumu1, Winnie A. Okeyo1, Elly O. Munde1, Evans Raballah2, Samwel B. Anyona1, Collins Ouma1
1 Maseno University, Maseno, Kenya

2 Masinde Muliro University of Science and Technology, Kakamega, Kenya

Overview

BackgroundIn malaria holoendemic areas such as western Kenya,
Plasmodium falciparum malaria is detected throughmicroscopic examination of blood films from asuspected patient. The use of an invasive procedure
to obtain blood as the primary source suffers frommany challenges, which include: the pain associatedwith a needle that has turned away many would-be
volunteers in clinical studies and the risk of
inoculating the study participants with pathogenic
microbes if proper sterilization is not performed at
the point of injection. Therefore, an immediate needto develop a non-invasive, specific and sensitive
method of detecting malaria and other diseases inroutine health care services is desirable. DNA has
been an important source of specimen in genetic
variants and disease association studies.

•MethodologyA cross-sectional study was conducted to evaluate thequality of saliva-derived DNA relative to traditional
blood derived DNA of the same individuals withclinical symptoms of malaria (aged ≥ 4 years; n= 100)and resident in Plasmodium falciparum holoendemictransmission region of western Kenya. Participants
provided matched blood and saliva samples
for microbial DNA using DNA Genotek’s
OMNIgene®•ORAL self-collection kit. The extracted
DNA was subsequently used to genotype parasite
Merozoite Surface Protein (MSP)-2. The MSP-2 parasite
genotyping was carried out through nested PCR.

•••

Globally, falciparum malaria accounts for thegreatest degree of malaria-related morbidity
and mortality (WHO, 2000).1
Traditional methods have always employed theuse of Giemsa-stained thin or thick blood films
in microscopy studies for detection of malaria
parasitemia (Lieke et al., 2004; Wongsrichanalai
et al., 2007).2, 3
Rapid diagnostic tests (RDT) or “dipstick”, anantigen-based detection method, is used to detect
protein markers of Plasmodium species in theblood of asymptomatic individuals. This approachstill requires blood and therefore remains invasive.
Taken together, these factors may reduce therapid response rate in management of malaria,
in the process posing a significant problem tomalaria surveillance and control. Thus, analternative source for malaria diagnosis thatincorporates molecular tools is a necessity.

PurposeTo evaluate the quality of saliva-derived DNA relative
to traditional blood derived DNA of the same
individuals with clinical symptoms of malaria inwestern Kenya.

Study site and methodology
Study design: The study was carried out in a rural
setting in western Kenya.
Individuals aged 4 years and above (n= 100) of bothgenders and presenting with clinical symptoms ofmalaria were recruited following written informed
consent from the parents/guardians of the study
participants.

For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.
PD-WP-00035 Issue 2/2015-06© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

Ethical approval: The study was approved by
the Maseno University Ethical Review Committee(MUERC).
Blood and saliva sample collection: Children
presenting with clinical symptoms of malaria in arural hospital setting in western Kenya were initially
screened for parasitemia via Giemsa-stained slides
and resolution on a microscope. All those that werepositive for parasitemia provided additional 100 µLof blood, which was blotted onto FTA Classic
cards (Whatman Inc., Clifton, NJ). Furthermore,
additional saliva samples were collected using
the OMNIgene•ORAL kit for collection ofmicrobial DNA (DNA Genotek, Ottawa, ON).
DNA extraction: The saliva samples collected wereextracted through the OMNIgene•ORAL protocols
as per manufacturer’s instructions (DNA Genotek,Ottawa, ON). Following complete drying of the FTAblood spots, DNA was extracted using the Puregene®
DNA extraction kit Gentra® system (Gentra Systems,Inc., Minneapolis, MN) according to themanufacturer’s instructions.
Quantification of DNA: DNA obtained from bloodand saliva was quantified using NanoDrop 2000
UVVis spectrophotometer (Thermo Scientific,Waltham, MA).
Genotyping of parasite MSP-2 variants:
Plasmodium falciparum genotyping was performedon blood and saliva DNA extracts using nested PCR
and MSP2 family-specific primers as previously
described (Snounou et al., 1999).4
Extraction
protocolGentra

DNA source:Blood

40.2 ± 8.4 ng/µL

DNA source:Saliva–OMNIgene•ORAL

56.3 ± 0.3 ng/µL

S= Pf PCR from OMNIgene•ORAL/saliva kit
O= Pf PCR using DNA obtained from blood
M= Molecular weight marker
Figure 1: PCR results demonstrate DNA from the OMNIgene•ORAL/
saliva kits performs as well as DNA from blood in detection ofcirculating Plasmodium falciparum parasites.

Conclusions••

DNA from the OMNIgene•ORAL/saliva kits
works the same as DNA from other sources(e.g., blood) in detection of circulating
Plasmodium falciparum parasites.
Future approaches should utilize saliva kits
to avoid invasive blood sample collection for
diagnosis and research.

Funding••

Pfizer Royal Society (Awarded to Profesor Collins Oumafor Best Scientist, 2010).
OMNIgene•ORAL kits obtained from DNA Genotek Inc.,
Canada.

References
1234WHO. 2000. World Malaria Report. WHO Press, Geneva
Switzerland.
Lieke et al., Infect Immun.2004; 72: 6817-6824.
Wongsrichanalai et al., Am J Trop Med. 2007; 77:119-127.
Snounou et al., Mol Biochem Parasitol. 1993; 61: 315-320.

Table 1: Mean DNA concentration from saliva and blood
The total DNA yield (from Gentra Systems and OMNIgene•ORAL/saliva kit) was estimated by UV absorption based upon themean A260/A280 nm ratio. DNA yield from blood and saliva kit
are comparable.
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®OMNIgene is a registered trademark of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.
PD-WP-00035 Issue 2/2015-06© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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