Saliva as a proven, non-invasive sample type for molecular malaria testingand surveillance using OMNIgene®•ORAL at ambient temperatures
David Visanuvimol

DNA Genotek, Ottawa, Ontario, Canada
2014-09-19

BackgroundThe 2013 World Malaria report estimated there
were 207 million malaria cases worldwide andan estimated 627,000 deaths in 2012.
On a global basis only 14% of malaria cases
are detected.
Countries with high malaria burden reported lower
case detection rates; 85% of estimated cases occur
in countries where surveillance systems are weakest.

Limitations of current diagnostic testing
Current methods of identifying malaria infections
are by antigen-based Rapid Diagnostic Tests (RDT’s)and microscopy. These methods are labor-intensive,
require supervision by healthcare workers andexamination of thin/thick blood smears by skilled
microscopists. Moreover, RDTs and microscopic
examinations do not allow the detection of low-level,
sub-patent malaria infections.
While RDT’s have been instrumental in improving
speed and precision of malaria diagnosis, the usageof blood and blood spots presents several barriersfor RDT’s, thereby impacting their adoption
as surveillance tools. These barriers include:•••••

pain associated with finger pricking
fear of contracting blood-borne diseases
proper disposal of used needles
physical damage and environmental
susceptibility with RDT test strips
need for supervision with RDTs to ensure
correct interpretation of results and adherence
to hygiene practices.

Moreover, while Plasmodium DNA can be extracted
from dried blood blotted onto Whatman papers,
poor extraction practice may result in crosscontamination and sub-standard quality andquantity of Plasmodium DNA. The obstacles
associated with using RDT’s and dried blood
blots impact the implementation of thesemethods for nationwide malaria surveillance
and monitoring programs.

Saliva as an alternative solution formalaria detection and surveillance
Saliva collection is a non-invasive alternative from
RDT’s for malaria detection and as a surveillance
tool. Both P. falciparum and P. vivax DNA have been
detected in saliva samples of malaria patients.1, 2, 3, 4
It has been demonstrated that 1 mL of whole saliva
samples harbours detectable levels of Plasmodium
spp. DNA for downstream sequencing of pfdhfr and
18S rRNA.5, 6 Therefore, saliva can be sampled forhigh sensitivity and specificity molecular-based
malaria diagnosis. Additionally, participant
compliance is increased with pain-free and easy
saliva collection; therefore, providing greater accessto Plasmodium DNA for improved monitoring ofmalaria transmission, identification of sub-patent ormixed Plasmodium species infections, and patient
screening in artemisinin-resistance-emerging
regions/elimination settings.

OMNIgene•ORAL for sample stability andtesting scalability
OMNIgene•ORAL enables non-invasive and painfree collection of Plasmodium spp. via saliva. TheOMNIgene•ORAL chemistry stabilizes Plasmodium
DNA in 1 mL of saliva at ambient temperature for up
to 1 year, eliminating the cost and complexity of cold
storage making it ideal for field collection in remoteand low-resource settings. The easy-to-use andreliable nature of the OMNIgene•ORAL kits
improves patient compliance in both adults andchildren.

For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.
PD-WP-00041 Issue 3/2015-02© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

The utility of OMNIgene•ORAL kits in combination
with highly sensitive PCR-based methods such as
nested PCR and LAMP assays will facilitate theadoption of molecular-based malaria detection
practices that will enable screening of samples from
populations of all ages and genders, including
high-risk groups such as pregnant women.
A saliva-based approach of Plasmodium DNA
collection that ensures integrity of parasitic DNAregardless of humidity and temperature caneﬀectively be scaled and integrated into nationalmalaria detection and surveillance programs that
require one centralized processing center and basic
laboratory infrastructure.

The following data is from ongoing pilot studies
that show promising indications for the useof OMNIgene•ORAL as a non-invasive alternativefor malaria diagnostics.
P. falciparum DNA from 1 mL of saliva is a reliablesample type for malaria detection (n=100)(Courtesy of Dr. Collins Ouma, Maseno University,Kenya, as presented at the 6th MIM Pan-AfricanMalaria Conference in Durban, South African).

Extraction
protocolGentraOMNIgene

DNA source:Blood

40.2 ± 8.4 ng/µL–DNA source:
Saliva–56.3 ± 0.3 ng/µL

Table 1: Mean DNA concentration from saliva and blood.
The total DNA yield (from Gentra Systems and OMNIgene/saliva kit)
was estimated by UV absorption based upon the mean A260/A280 nm
ratio. DNA yield from blood and saliva kit are comparable.

“I recommend that future approaches should utilize
OMNIgene•ORAL self collection kits to avoid invasive
sample collections, improve patient recruitment,improve the patient experience, and enhance malaria
diagnostics and research.”- Dr. Collins Ouma

1 mL of saliva stabilized in OMNIgene•ORAL candetect P. falciparum in symptomatic patients
ranging from 6,000-70,000 parasites/µL (n=6)(Courtesy of Dr. Deus Ishengoma, National Malaria
Research Council, Tanzania).
13 12 11 10 9

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

87654

32

1

Interpretations key
1. 21179 (saliva sample)
9. Positive P. falciparum control Pf (3D7)
2. 21166 (saliva sample)
10. Positive P. falciparum control (FCR3)
3. 21048 (saliva sample)
11. P. falciparum known sample
4. 21355 (saliva sample)
12. Positive P.malariae control
5. 21165 (saliva sample)
13. Negative control (ddH20)
6. 21356 (saliva sample)
14. 50 bp ladder marker
7. Negative control
8. Negative control
Figure 2: Agarose gel electrophoresis results of samples and control
from PCR products.

ID21179
21166
21048
21355
21165
21356

Temp. (OC)
37.3
38.3
37.8
39.0
37.6
37.9

Microscopy
0/100
Pf.313/200
0/100
Pf.1744/200
Pf.158/200
Pf.1064/200

mRDT
Negative
Positive
Negative
Positive
Positive
Positive

PCR
Negative
Negative
Negative
Positive
Positive
Positive

Table 2: Patient information, microscopy , mRDT and PCR results.
For research use only, not for use in diagnostic procedures.
Not available for clinical diagnostic use in the United States.
PD-WP-00041 Issue 3/2015-02© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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“The OMNIgene•ORAL kit proved successful in the
majority of the tested samples with comparable
results as detected by mRDT and microscopy.
Therefore, the non-invasive OMNIgene•ORAL
kit offers potential for use in malaria diagnosis.”- Dr. Deus Ishengoma

P. falciparum DNA collected from febrile
participants shows high specificity and sensitivity
compared to blood (n=11)(Courtesy of Dr. Kenji Obadiah Mfuh,
John A. Burns School of Medicine,
University of Hawaii at Manoa, USA)“We have successfully extracted P. falciparum fromthe OM-501 kits. Parasite genetic diversity using
MSP1 and MSP2 allelic families was also determined.
I’m happy to say that we have sufficient amount ofDNA for genetic analysis from the kits (n=69).”- Dr. Magatte Ndiaye, Université Cheikh Anta Diop
(University of Dakar), Senegal on the use of OM‑501
for temporal dynamics of molecular markersof anti‑malarial drug resistance in P. falciparum
parasite populations in Senegal.

Conclusions

120.00%100.00%80.00%
60.00%
40.00%

Saliva PCR

20.00%RDT results

0.00%

Additional testimony:

Saliva PCR

Specificity
87.50%

Sensitivity
100%

PPV
75%

NPV
100%

RDT results

25%

100%

33.30%

100%

Given that only 14% of malaria cases are detected
globally and several limitations exist to current
diagnostic testing methods, there is a need toexplore other approaches that enhance malaria
diagnostics and national surveillance programs.
OMNIgene•ORAL is a saliva-based collection andstabilization kit that allows for the non-invasive,proactive sampling and storage of Plasmodium
DNA at ambient temperature.

“Malaria parasite DNA is present in the saliva ofinfected individuals and that saliva can be used
as an alternative for non-invasive sample for thediagnosis of malaria in a PCR-based reaction.
This pilot study also proves that the OMNIgene•ORAL
kit is very effective in preserving malaria DNA atroom temperature.”- Dr. Kenji Obadiah Mfuh

References
12
3
4
5
6

Putaporntip et al. Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial
DNA-based PCR assays. Clin Microbiol Infect 17: 1484–1491 (2011)
Nwakanma et al. Quantitative Detection of Plasmodium falciparum DNA in Saliva, Blood, and Urine. J Infect Dis 199:1567–74 (2009)
Gbotosho et al. Rapid Detection of Lactate Dehydrogenase and Genotyping of Plasmodium falciparum in Saliva of Children withAcute Uncomplicated Malaria. Am J Trop Med Hyg 83: 496–501 (2010)
Singh et al. Rapid detection of Plasmodium vivax in saliva and blood using loop mediated isothermal amplification (LAMP) assay.
J Infect 67:245-247 (2013)
Ongagna-Yhombi et al. Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative
lateral flow analysis. Malaria Journal 12:74 (2013)
Singh et al. Comparison of three PCR-based assays for the non-invasive diagnosis of malaria: detection of Plasmodium parasites
in blood and saliva. Eur J Clin Microbiol Infect Dis 33:1631–1639 (2014Some DNA Genotek products may not be available in all geographic regions.
®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-00041 Issue 3/2015-02© 2015 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

www.dnagenotek.com • support@dnagenotek.com

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