screen for malaria carrier infections using human saliva
Ofentse J. Pooe1, Addmore Shonhai1 and Sungano
of Biochemistry and Microbiology, University of Zululand,
Private Bag 1001,
KwaDlangezwa, 3886, South
Malaria Research Trust, P. O. Box 630166, Choma, Zambia.
of Molecular Microbiology and Immunology, Johns Hopkins
Bloomberg School of Public Health,
615 N. Wolfe Street, Baltimore MD21205, USA.
*Corresponding author. E-mail:
Accepted 9 November, 2011
With endemic countries now aiming for elimination, the
detection of malaria infections, with or without symptoms,
is increasingly important for monitoring and evaluation
programmes. Current malaria screening methods
necessitate blood withdrawal. This invasive approach is
constrained, especially for identifying the asymptomatic
carrier reservoir, since segments of communities with blood
taboos avoid participating. Proof of concept has previously
been shown for molecular detection of malaria infection
using human saliva samples. The current study optimized
saliva-based malaria detection in an area of southern
Zambia. Saliva pellet fractions proved a more reliable
source of amplifiable parasite DNA compared to the soluble
fraction. After optimizing DNA extraction and amplification,
saliva-based PCR showed 94.1% sensitivity and 97%
specificity, using nested PCR on blood samples as gold
standard. This study demonstrates that saliva samples are a
reliable non-invasive alternative to blood for the PCR
detection of asymptomatic and submicroscopic malaria
Malaria, saliva, polymerase chain reaction.
Malaria remains a major killer disease accounting for nearly
800,000 deaths in 2009 (WHO, 2010). The deve-lopment of
accurate diagnostic tests that are appropriate for use in
under-developed countries ravaged by malaria is important.
Traditional diagnostic methods such as microscopy are beset
with limitations. For example, experienced microscopists
tend to detect malaria at a density not less than 500
parasites/μl in routine laboratory tests (Milne et al.,
1994). The advent of polymerase chain reaction (PCR) based
methods for the detection of malaria has seen a huge
improvement in malaria detection levels 1000 times higher
than data from microscopy (Schoone et al., 2000). Indeed,
PCR conducted on blood samples also reportedly detects the
parasite 5 days before the parasite can be detected by
experienced microscopists (Andrews et al., 2005). Some of
the disadvantages of PCR based methods in malaria diagnosis
include risk of contamination, the requirement of a thermal
cycler and electricity to conduct the test. However, the
advent of loop-mediated isothermal amplification (LAMP)
which has capability of amplifying DNA at a constant
temperature holds promise as a useful field technique for
application in the diagnosis of malaria (Polley et al.,
The use of blood in the diagnosis of malaria is problematic
due to several factors. Amongst these is the fact that
infected erythrocytes tend to be sequestered away from
circulation at certain stages in the develop-ment of malaria
(Delley et al., 2000). Further-more, drawing of blood for
clinical purposes is fraught with challenges as this
procedure must be conducted by skilled personnel who are not
always readily available in remote locations of most
countries affected by malaria. In addition, subjects tend to
shy away from donating blood for clinical purposes due
to fear of accidentally contracting infectious diseases
such as HIV/AIDS and in some cultures donating blood is a
cultural and religious taboo. As endemic countries are
moving towards malaria elimination, this poses a major
constraint, since measure-ment of malaria infections, with
or without symptoms is now increasingly important in
monitoring and evaluation programmes.
Blood-based malaria testing approaches tend to miss
asymptomatic, submicroscopic infections that are capable of
sustaining transmission (Schneider et al., 2007; Gahutu et
al., 2011), since individuals that are not sick are even
less likely to participate in invasive surveys.
For this reason, several studies have of late been focusing
on detecting malaria in saliva, urine and surface mucosa by
amplifying parasite DNA in these fluids (Mharakurwa et al.,
2006; Nwakanma et al., 2009;
et al., 2010).
Data based on PCR conducted on saliva show more reliability
in malaria detection than tests conducted on urine samples (Mharakurwa
et al., 2006; Nwakanma et al., 2009). Saliva is a fluid that
occurs in the oral cavity and is made up of numerous
constituents, mostly secretary products from various sources
such as salivary glands, as well as blood-derived compounds
(Lima et al., 2010). Water constitutes 90% of saliva, and
the content of the dissolved components in saliva vary from
time to time in the same individual and indeed these
constituents vary across individuals. As testimony to its
richness in organic constituents, saliva is known to contain
at least 400 proteins (Lima et al., 2010). The use of saliva
as material for disease diagnosis lends itself to the fact
that saliva glands occur at interfaces that connect them
with blood vessels, thus facilitating exchange of materials
with the circulatory system.
Therefore, it is not surprising that residues of material
from human pathogens end up in saliva. One of the first
studies to demonstrate the availability of DNA from human
pathogens in saliva involved the detection of hepatitis B
virus DNA in saliva at a concentration comparable to levels
in blood (van der Eijk et al., 2004). It is still unknown
how DNA from malaria parasites ends up in saliva. It is
further unclear whether whole parasite cells debris or
parasite cells end up in saliva or whether it is merely
parasite DNA that diffuses from blood into human saliva. In
addition, the reliability of PCR as a tool for the detection
of malaria in saliva needs to be optimized and to be
demonstrated to work consistently across various
Furthermore, the conditions under which the saliva samples
are stored from point of source seem to influence the
sensitivity of the test (Buppan et al., 2010). Additionally,
inconsistent data where PCR conducted on malaria DNA derived
from saliva, urine and buccal mucosa could not be used to
distinguish between mixed parasites that were confirmed
present by PCR conducted on parasite DNA derived from blood
et al., 2010). It has previously been
suggested that the sensitivity of PCR for the detection of
malaria in saliva could be improved by using primers
targeting short amplicons (300 bp or less) as the DNA
template is less prone to degradation (Mharakurwa et al.,
This study sought to establish the optimal fraction of
saliva (between the pellet and soluble fractions) for
sourcing reliable template DNA for use in PCR. Furthermore,
the study also established whether shorter amplicon primers
targeting the ‘pfdhfr’ gene (Mharakurwa et al., 2006) would
yield different PCR detection sensitivity to longer amplicon
Materials and Methods
Study area and population
The study samples were collected from the vicinity of Macha,
located in the Zambian southern province. Natural malaria
transmission at Macha is hyperendemic, the major vectors being
Anopheles arabiensis and Anopheles funestus. In
the study area the collection of blood samples during
major malaria surveys or research, frequently led to tension and
reduced community participation, due to recurrent suspicion,
usually about Satanism (associated with blood). The development
of simple bloodless alternative sampling approaches would lead
to greater community participation for research and control of
Samples used in this study were collected during peak malaria
transmission seasons between year 2008 and 2009. Willing
participants from headmen areas in the vicinity of Macha were
screened for malaria by microscopy. A total of 88
were recruited for this study, 41% males and
59% females with an age range between 3 months and 99 years
(mean = 29.6 years; median = 18 years), following full
explanation of the objectives, procedures, risks and benefits of
the study. In the case of young children, informed consent was
sought from the parents or guardians. The study excluded
individuals with severe and complicated malaria or complicated
medical conditions as recommended (WHO, 2003). Data collection
was conducted using structured questionnaires. Axillary
temperature was taken to monitor if the participants had
pyrexia. The history of any disease symptoms and drug intake in
the past 48 h for all adult participants were recorded. For
active recruitment, only individuals resident in the selected
headman area were enrolled. In passive enrolment, only
individuals living within walking distance from the hospital,
who were able and willing to return for study follow-up were
included. Thick films and filter paper (Whatman ® No 3 MM) blood
blots were collected. Microscopic slides were examined at MIAM
by an experienced microscopist followed by feedback to the
community and treatment for confirmed malaria cases. On the day
of feedback, whole saliva samples (5 ml) was collected from all
willing positive and negative individuals in sterile tubes.
The saliva specimens were later aliquoted into 1 ml replicate
amounts in microcentrifuge tubes at the laboratory and either
immediately extracted or stored at -20°C for later extractions
Whole saliva samples of 500 μl were centrifuged in 1.5 ml
microcentrifuge tube for 3 min at 20,000 g. The entire soluble
saliva fraction was aspirated out without disturbing the pellet;
the supernatant was collected into a new sterile tube. DNA was
extracted from the pellet fraction and the soluble fraction of
saliva respectively using the crude cell lysates protocol
following the manufacturer's (Qiagen DNeasy® purification kit)
instructions. The final DNA elution was carried out in 50 μl
volumes. Blood spots collected on filter paper were extracted
using the Chelex method (Plowe et al., 1995) ADDIN EN.CITE
ADDIN EN.CITE.DATA .
Nested PCR amplification
was detected from blood and saliva DNA extracts by nested PCR
following a previously described procedure (Mharakurwa et al., 2006.
Briefly all PCR ‘pfdhfr’ amplifications consisted of initial
denaturation at 94°C for 2 min, followed by 25 cycles of
denaturation at 94°C for 45 s, annealing at 43.4°C for 45 s and
extension at 65°C for 1 min. The final extension step was at 65°C
for 2 min. Both primary and secondary PCR reactions comprised 1.2 μl
template, 0.25 μM primers, 1.5 mM magnesium chloride, 200 μM dNTP's,
1X PCR Buffer and 0.6 U of Taq DNA polymerase in 15 μl volumes. Two
sets of PCR primers were used that define fragments on the ‘pfdhfr’
domain. The first set were standard primers (M1[3..23]:
for the primary round (643 bp product), followed by (F [144..172]:
for the secondary amplification (326 bp product) as described by
Duraisingh et al. (1998). The second set of primers, defining
shorter amplicon fragments were (U1[121..143]:
for the primary round (273 bp product), followed by (U3 [144..172]:
for the secondary amplification (229 bp product).
The PCR amplifications were performed in a Thermo Electron® PX2
(HBPX2) thermal cycler. The PCR amplicons obtained were resolved by
electrophoresis on 1.5% agarose gels stained in ethidium bromide and
visualised by UV transillumination on a 1D Kodak (EDAS 290) imaging
Diagnostic performance was measured by calculating sensitivity,
speciﬁcity, positive predictive values (PPV), negative predictive
values (NPV) and receiver operating characteristic (ROC) area under
the curve (AUC) calculated using MedCalc ® version 184.108.40.206 (MedCalc
Software, Mariakerke, Belgium). Pearson’s chi-square (c2)
test of association was used to evaluate the strength of association
between various tests. The degree of agreement was interpreted as
follows: poor (<0.20) to very good (0.81 to 1) based on Kappa
interpretation (Cohen, 1968).
The study was approved by University of Zambia research ethics
committee and University of Zululand ethics committee. Study
permission was sought from local chiefs and headmen in whose area
the study was conducted. Patient participation was obtained through
the consent of the patients themselves or guardians where minors
A total of 88 samples (blood and
saliva) were collected and analyzed
from willing individuals who
participated in this study. The age
range was from 3 months to 99 years
old (mean = 29.6 years; median = 18
years). All the participants of this
study were asymptomatic, none of the
examined volunteers had a body
temperature reading above 38°C nor
was there any below 35°C on the day
When the blood extracts were
PCR amplification with the long
amplicon primers, a total of 33
(37.5%) samples were confirmed to be
P. falciparum positive. As
could be expected, PCR had a higher
detection sensitivity than
microscopy which confirmed only 13
(14.7%) positives. However, short
amplicon primers were able to detect
51 (57.95%) P. falciparum
infections, 31 (35.23%) were
submicroscopic and 18 (20.45%) were
below long amplicon primer detection
limit. Short amplicon primers were
more sensitive (95%) as compared to
long amplicon primers (52.63%) in
amplifying P. falciparum DNA,
using microscopy results as
AUC and ROC curve analysis were
conducted to determine the visual
indices for the accuracy of
conducting PCR using the long
primers compared to the short
primers (Figure 2). The further the
curve lies above the diagonal
reference line, the greater the
accuracy of the primer set used, the
short amplicon primer set curve lies
the furthest from the reference line
as compared to the long amplicon
primer set curve (Figure 2). Short
amplicon primers had a greater AUC
and therefore were more accurate
when compared to long amplicon
primers; 0.740, p = 0.0005 and
0.594, p = 0.2176 respectively.
These results support claims
previously noted by Mharakurwa et
al. (2006) that short amplicon
primers enhance the sensitivity for
P. falciparum DNA
Human saliva can be used as a source
of amplifiable malaria parasite DNA
in infected patients as noted by
previous investigators (Mharakurwa
et al., 2006; Nwakanma et al., 2009;
et al., 2010). To determine the
fraction of saliva harbouring the
malaria amplifiable DNA, saliva
samples obtained from the 42 P.
falciparum positive human
and three negative
all samples where separated into two
distinct fractions, the soluble and
insoluble fractions. After
centrifugation, DNA was extracted
from the saliva insoluble and
soluble fractions and then subjected
to nested PCR in separate batches.
The amplified products were resolved
by gel electrophoresis (Figure 3).
The short amplicon primers were
subsequently used for all PCR
amplifications. Furthermore, we
separated the pellet fraction of
saliva from the soluble fraction. We
then purified DNA from these
fractions using the Qiagen kit. DNA
amplified from the pellet only
fraction had higher amplification
success than DNA isolated from the
soluble fraction of saliva. The
sensitivity and specificity values
achieved using DNA derived from
saliva pellet fraction compared to
DNA isolated from blood were 94.12
and 97.3%, respectively (Table 1).
Diagnostic performance comparing
nested PCR results for saliva with
blood samples as reference standard.
PCR on DNA derived from
Positive predictive value
Negative predictive value
Kappa value (κ)
PCR is expected to
generally have an elevated sensitivity when compared
to microscopy especially in detecting parasitemia at
levels undetectable to microscopy (Snounou et al.,
1993). Results obtained from PCR amplification on
blood samples were superior to those obtained by
thick film microscopy. Some of the samples that were
thick film-negative were reported to be positive by
PCR for P. falciparum infection. In the current
study at least 35.23% of the samples were reported
as positive for malaria infection using PCR (based
on short amplicon primers), compared to 14.7%
reported as positive using microscopy. A similar
finding was reported where microscopy detected a
total of 350 P. falciparum infections while PCR
detected a further 331 P. falciparum submicroscopic
infections (Zurovac et al., 2006). According to
Bejon et al. (2006), true parasite counts are likely
to be under-estimated by microscopy due to the
staining procedure, which may account for poor
micro-scopy performance. In order for endemic
countries to effectively manage malaria
interventions, asymptomatic infection and latent
disease reservoirs need to be closely monitored so
as to avoid potential resurgences. The nested PCR
diagnostic method can be useful for detection of
latent malaria carrier infections. As previously
proposed by Mharakurwa et al. (2006), short amplicon
primers used in this study had a higher
amplification rate as compared to long amplicon
primers targeting the same region on the ‘pfdhfr’
domain. Consequently, the former were more reliable
for detecting latent infections. It might be
possible that at low level of parasite density, long
amplicon primers were less reliable than short
amplicon primers because of the poor recovery of
good quality parasite DNA.
Parasite DNA may be degraded during storage and transportation of
samples leading to low amplification sensitivity
when using long amplicon primers. In addition, the
acidity of the buccal acidity may also promote
degradation of parasite DNA, leading to poor
amplicon yields. However, mere primer annealing
properties may also lead to such differences,
regardless of amplicon size.
We further spun the saliva to obtain pellet and soluble fractions.
We sought to understand which of the two fractions
of saliva would provide better quality of
amplifiable parasite DNA. Based on the nested PCR
findings, DNA extracted from the pellet fraction was
a more reliable template for the PCR test. It is
possible that P. falciparum DNA is introduced into
human saliva through ruptured RBCs or DNA from
parasites trapped in macrophages (Kaufman and
Lamster, 2002). The DNA amplified in the supernatant
portion of saliva may be due to trace amounts of
pellet material taken up with supernatant during
sample preparation and extraction. However, further
study is still required to clarify how malarial DNA
is transported to saliva of malaria infected
patients. Through optimising the nested PCR recipe
on saliva DNA extracts, this study was able to
achieve high amplicon yields than previously
reported (Buppan et al., 2010), relative to DNA
purified from blood samples. In this study, DNA
derived from human saliva samples using the Qiagen
extraction kit displayed 94% sensitivity and 97%
specificity, using regular PCR on blood as the gold
standard. Buppan et al. (2010), suggests that saliva
samples preserved in ethanol yielded superior
positive PCR results when compared to samples kept
on ice. However, the absence of ethanol preservation
in this study does not appear to have negatively
affected PCR amplification. A very good agreement (κ
= 0.907) was observed for DNA derived from saliva
using the Qiagen extraction kit relative to DNA
purified from blood samples. This study confirms the
reliability of saliva as an alternative source for
malaria infection diagnosis which may be adopted for
large scale malaria screenings (Breman et al.,
2004). In this study, only 15.9% of the volunteers
had a history of malaria-related symptoms. Thus,
under normal circumstances the 84.1% asymptomatic
patients would not have gone for malaria testing,
since they did not experience any need to seek
To progress towards the goal of effective malaria control and
eventual elimination, the accurate identification of
asymptomatic parasite carriers who are often sources
of perpetual disease transmission is vital. The use
of non-invasive saliva-based screening affords an
accurate and fundamentally more pragmatic approach
for maximizing community participation, especially
in detecting foci of asymptomatic reservoir
infections. Obviating the use of sharps or needles,
blood drawing and associated community taboos,
healthy infection carriers and vulnerable groups
alike are far more likely to cooperate in surveys
for operational research and control programmes.
Our study shows an accurate and reliable method for the PCR
detection of malaria using saliva samples as an alternative
to blood samples. This non-invasive approach affords
fundamental practical advantages on safety, community
participation and minimizing bias in large community surveys
with or without need for repeated testing such as drug or
vaccine efficacy trials. Future studies could examine the
use of loop-mediated isothermal amplification (LAMP) and
exploring possibilities of migrating from the bench-top to a
point-of-care device such as a biosensor chip. This would
introduce sensitive tools for surveillance and targeting of
asymptomatic reservoirs of infection in malaria control and
We would like to express special gratitude to the
communities, the headman and chiefs in the vicinity of Macha,
Zambia for their participation in this study. We would also
like to thank Mr. Cliff Sing'anga, Ms. Mwiche Siame, Mr.
Gift Moono and the rest of the staff at Macha Research Trust
for their hospitality and assistance. We are grateful to Mr.
Edgar Jembere, Department of Computer Science and University
of Zululand for technical support. This study was supported
by the Johns Hopkins Malaria Research Institute. OJP
received funding from the University of Zululand Research
Committee and a scholarship from the South African National
Research Foundation which enabled him to conduct this study.
A-Elgayoum SME, El-Rayah E-A, Giha HA (2010).
Towards a noninvasive approach to malaria diagnosis: detection
of parasite DNA in body secretions and surface mucosa.
J. Mol. Microbiol. Biotechnol., 18(3): 148-155.
Andrews L, Andersen RF, Webster D, Dunachie S, Walther RM, Bejon
P, Hunt-Cooke A, Bergson G, Sanderson F, Hill AV, Gilbert SC
(2005). Quantitative real-time polymerase chain reaction
for malaria diagnosis and its use in malaria vaccine clinical
trials. Am. J. Trop. Med. Hyg., 73(1): 191-198.
Bejon P, Andrews L, Hunt-Cooke A, Sanderson F, Gilbert SC, Hill
AVS (2006). Thick blood film examination for Plasmodium
falciparum malaria has reduced sensitivity and
underestimates parasite density.
Malar J., 5: 104-107.
Breman JG, Alilio MS, Mills A (2004).
Conquering the intolerable burden of malaria: what's new, what's
needed: a summary. Am. J. Trop. Med. Hyg., 71(2): 1-15.
Buppan P, Putaporntip C, Pattanawong USS, Jongwutiwes S (2010).
Comparative detection of Plasmodium vivax and
Plasmodium falciparum DNA in saliva and urine samples from
symptomatic malaria patients in a low endemic area.
Malar J., 9: 72-78.
Cohen J (1968). Weighted kappa: nominal scale agreement with
provision for scaled disagreement or partial credit. Psychol.
Bull., 70(4): 213-220.
Delley V, Bouvier P, Breslow N, Doumbo O, Sagara I, Diakite M,
Mauris A, Dolo A, Rougemont A (2000).
What does a single determination of malaria parasite density
mean? A longitudinal survey in Mali. Trop. Med. Int. Health,
Duraisingh MT, Curtis J, Warhurst DC
(1998). Plasmodium falciparum: detection of
polymorphisms in the dihydrofolate reductase and dihydropteroate
synthetase genes by PCR and restriction digestion. Exp.
JB, Steininger C, Shyirambere C, Zeile I, Cwinya-Ay N, Danquah
I, Larsen CH, Eggelte TA, Uwimana A, Karema C, Musemakweri A,
Harms G, Mockenhaupt FP (2011). Prevalence and risk
factors of malaria among children in southern highland Rwanda.
Malar J., 10: 134-145.
Kaufman E, Lamster IB (2002). The diagnostic applications of
saliva - a review. Crit. Rev. Oral Bio. Med., 13(2): 197-212.
Lima DP, Diniz DG, Moimaz SA, Sumida DH, Okamoto AC
(2010). Saliva: reflection of the body. Int. J. Infect.
Dis., 14(3): 184-188.
Mharakurwa S, Simoloka C, Thuma PE, Shiff CJ, Sullivan DJ
(2006). PCR detection of Plasmodium falciparum in
human urine and saliva samples.
Malar J., 5: 103-109.
Milne LM, Kyi MS, Chiodini PL, Warhurst DC (1994).
Accuracy of routine laboratory diagnosis of malaria in the
United Kingdom. J. Clin. Pathol., 47(8): 740-742.
Nwakanma DC, Gomez-Escobar N, Walther M, Crozier S, Dubovsky F,
Malkin E, Locke E, Conway DJ (2009). Quantitative detection of
Plasmodium falciparum DNA in saliva, blood, and urine. J.
Infect. Dis., 199(11): 1567-1574.
Plowe CV, Djimde A, Bouare M, Doumbo O, Wellems TE (1995).
Pyrimethamine and proguanil resistance-conferring mutations in
Plasmodium falciparum dihydrofolate reductase: polymerase
chain reaction methods for surveillance in Africa. Am. J. Trop.
Med. Hyg., 52(6): 565-568.
Polley SD, Mori Y, Watson J, Perkins MD, Gonzalez IJ, Notomi T,
Chiodini PL, Sutherland CJ (2010).
Mitochondrial DNA targets increase sensitivity of malaria
detection using loop-mediated isothermal amplification. J. Clin.
Microbiol., 48(8): 2866-2871.
Schneider P, Bousema JT, Gouagna LC, Otieno S, van de
Vegte-Bolmer M, Omar SA, Sauerwein RW (2007). Submicroscopic
Plasmodium falciparum gametocyte densities frequently result
in mosquito infection. Am. J. Trop. Med. Hyg., 76(3): 470-474.
Schoone GJ, Oskam L, Kroon NC, Schallig HD, Omar SA (2000).
Detection and quantification of Plasmodium falciparum in
blood samples using quantitative nucleic acid sequence-based
amplification. J. Clin. Microbiol., 38(11): 4072-4075.
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do
Rosario VE, Thaithong S, Brown KN
(1993). High sensitivity of detection of human malaria
parasites by the use of nested polymerase chain reaction. Mol.
Biochem. Parasitol., 61(2): 315-320.
van der Eijk AA, Niesters HG, Götz HM, Janssen HL, Schalm SW,
Osterhaus AD, de Man RA (2004).
Paired measurements of quantitative hepatitis B virus DNA in
saliva and serum of chronic hepatitis B patients: implications
for saliva as infectious agent.
J. Clin. Virol., 29(2): 92-94.
World Health Organization (WHO) Report (2003). Assessment and
Monitoring of Antimalarial drug efficacy for the treatment of
uncomplicated falciparum malaria. Geneva.
World Health Organization (WHO) report (2010). World Malaria
Zurovac D, Midia B, Ochola SA, English M, Snow RW
(2006). Microscopy and outpatient malaria case management
among older children and adults in Kenya. Trop. Med. Int.
Health, 11(4): 432-440.