JOURNAL OF CLINICAL MICROBIOLOGY, July 2011, p. 2411–2418 Vol. 49, No.

7
0095-1137/11/$12.00 doi:10.1128/JCM.02603-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Applied Genomics: Data Mining Reveals Species-Specific Malaria

Diagnostic Targets More Sensitive than 18S rRNA䌤†‡
Allison Demas,1,2,3#§ Jenna Oberstaller,4# Jeremy DeBarry,5# Naomi W. Lucchi,1,2
Ganesh Srinivasamoorthy,5 Deborah Sumari,6 Abdunoor M. Kabanywanyi,6 Leopoldo Villegas,7
Ananias A. Escalante,8 S. Patrick Kachur,1 John W. Barnwell,1 David S. Peterson,5,9
Venkatachalam Udhayakumar,1 and Jessica C. Kissinger4,5,10*
Malaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention,
Atlanta, Georgia1; Atlanta Research and Education Foundation/VA Medical Center, Decatur, Georgia2; Association of
Public Health Laboratories, Silver Spring, Maryland3; Department of Genetics4 and Center for Tropical and
Emerging Global Diseases,5 University of Georgia, Athens, Georgia; Ifakara Health Institute, Dar-es-Salaam,
Tanzania6; Asociación Civil Impacto Social, Tumeremo, Venezuela7; Arizona State University, Tempe,
Arizona8; Department of Infectious Disease, University of Georgia, Athens, Georgia9; and
Institute of Bioinformatics, University of Georgia, Athens, Georgia10
Received 22 December 2010/Returned for modification 9 February 2011/Accepted 18 April 2011

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treat-
ment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting
Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available
whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still
rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays.
However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of
nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and
amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax
to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We
show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when
utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/␮l. The reaction can
be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P.
falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from
Venezuela, and we have verified a sensitivity and specificity of ⬃100% for both targets compared with a nested
18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel
diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a
powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional
or multiplex PCR platforms.

Malaria continues to be a leading cause of morbidity and dium vivax, which also causes significant morbidity and some
mortality worldwide. It is responsible for 200,000 to 300,000 mortality (2, 10, 14, 23, 29, 38). P. falciparum and P. vivax also
diagnosed cases and 600,000 to 900,000 deaths in 2009 alone have wider global distributions than other species. The remain-
(40). Early detection and accurate diagnosis are the best tools ing three species (which are not the subject of this paper), P.
for saving lives in regions of endemicity. Correct species iden- malariae, P. ovale, and P. knowlesi, have different global distri-
tification and accurate diagnosis of mixed infections are of butions (with P. malariae being found primarily in South
particular importance for proper treatment in regions where America and Asia and P. ovale and P. knowlesi being found
multiple parasite species are endemic. Of the five species primarily in Asia) and different levels of morbidity and mor-
within the genus Plasmodium known to infect humans, Plas- tality.
modium falciparum is the most deadly, followed by Plasmo- Light microscopy remains the gold standard of malaria
diagnosis in regions of endemicity. While microscopy is cost-
effective and requires little equipment, a well-trained mi-
* Corresponding author. Mailing address: Center for Tropical and croscopist is essential. A highly trained and experienced
Emerging Global Diseases, Paul Coverdell Center, Rm. 370, 500 D.W. microscopist can typically detect parasitemias of as low as 90
Brooks Drive, University of Georgia, Athens, GA 30602. Phone: (706) to 200 parasites/␮l. Misdiagnosis may still occur due to low
542-6562. Fax: (706) 542-3585. E-mail: jkissing@uga.edu.
# These authors contributed equally to this work.
parasitemia or mixed infection. Immunochromatographic
§ Present address: School of Public Health, Harvard University, rapid diagnostic tests (RDTs) are increasingly being imple-
Cambridge, MA. mented in case management and control programs. RDTs
† Supplemental material for this article may be found at http://jcm identify the parasite antigens HRP2, pLDH, and pAldolase
.asm.org/.
䌤
Published ahead of print on 27 April 2011.
and may be pan-specific (for all Plasmodium species), P.
‡ The authors have paid a fee to allow immediate free access to falciparum specific, or both, depending on the test. RDTs
this article. are not effective for the full diagnosis of mixed infections, as

2411
2412 DEMAS ET AL. J. CLIN. MICROBIOL.

they can only distinguish P. falciparum and indicate the number, a low target copy number limits the detection ca-
presence or absence of another Plasmodium species. While pabilities of these assays, especially if the parasitemia is low.
they can detect parasitemia at levels as low as 100 parasites/ The 18S rRNA gene target also presents challenges for ef-
␮l, they are not quantitative (21). Additionally, the HRP2 fective multiplex platforms. The design of multiple primers to
antigen can persist in blood after parasite clearance, leading the same target can result in primer competition and decrease
to false-positive diagnoses. It has also been reported that up the efficiency of the assay. While multiplex assays for simulta-
to 40% of P. falciparum parasites in some parts of South neous detection of malaria parasite species do exist (25, 31,
America have HRP-2 gene deletions, increasing concerns 37), they show decreased sensitivity, particularly in detecting
about false-negative diagnoses (8). the minor species (20). Rubio et al. (31) designed a seminested
The use of molecular diagnostic tools is the most accurate two-tube multiplex PCR, with an initial genus-specific ampli-
and sensitive method for detecting malaria parasite species. fication followed by a secondary amplification using a universal
Their current use, however, is restricted to reference labora- Plasmodium primer and species-specific reverse primers. Pad-
tories or research studies, since there are limitations associated ley et al. (25) designed a one-tube multiplex assay, using spe-
with the use of molecular tools in regions of endemicity for cies-specific primers. However, both of these methods have
routine diagnostic use (including infrastructure problems, pro- been shown to perform less effectively than the standard
hibitive costs, a refrigerated or frozen supply cold chain, and nested PCR method (20). Taylor et al. (37) designed a multi-
the requirement for trained personnel). Despite these limita- plex real-time platform, relying on the increased sensitivity of
tions, molecular methods are the best methods for detecting both novel targets and fluorescent probes. However, this assay
multiple species and subclinical infections (4, 7), making them was most effective in duplex format and not as a true four-
invaluable for malaria parasite detection. Molecular methods species multiplex assay.
will become increasingly important given the proposed eradi- To address the limitations of existing molecular diagnostic
cation/elimination goals and the need to detect subclinical tools, we have mined Plasmodium genome sequence data and
infections (12). identified new target DNA sequences for improved molecular
PCR-based amplification methods, including multiplex diagnostic applications. Here we detail the method used to
PCR, real-time PCR, and, more recently, the loop-mediated identify these targets in P. falciparum and P. vivax, and we show
DNA amplification method (LAMP), have been developed to that they provide increased sensitivity in a single-step PCR and
detect malaria parasite species (11, 24, 25, 30, 31, 35, 37). increased efficacy in multiplex assays.
Molecular methods offer the advantage of highly specific dif-
ferentiation of Plasmodium species. Recently, molecular tech- MATERIALS AND METHODS
niques confirmed the natural infection of humans with the
Data harvesting. Assembled genome sequence data for P. falciparum (3D7
zoonotic P. knowlesi in Southeast Asia (33). This simian ma- strain) and P. vivax (Sal-1 strain) were obtained from PlasmoDB (release 5.5).
laria parasite species had not previously been found in humans The P. falciparum genome data consist of 14 sequences (23,264,338 bp), and the
in great numbers, and a similar morphology resulted in an P. vivax genome data consist of 2,747 sequences (27,007,990 bp). Differences in
incorrect P. malariae diagnosis by microscopy. the numbers of sequences between species reflect the more advanced state of P.
falciparum genome assembly relative to P. vivax. There are 14 highly assembled
The most widely used molecular target for the detection
chromosomes for each species and 2,733 unassigned contigs for P. vivax.
of Plasmodium and diagnosis of malaria was developed prior CRS screens and copy number determination. The pipeline shown in Fig. 1
to the completion of any Plasmodium genome sequence. and described below was constructed using custom PERL scripts. Repeat-
The target is the 18S rRNA gene(s) (11, 16, 30, 32, 34). This Scout (version 1.0.5, default parameters) (28) was used to identify genomic
target was a logical choice given its high sequence conser- consensus repeat sequences (CRS). Totals of 418 P. falciparum and 428 P.
vivax CRS were generated. The Tandem Repeat Finder program (TRF)
vation, the availability of universal primer sequences for its version 4.0 (3) was used to eliminate CRS with internal tandem repeats that
amplification, and the fact that it was known to exist in could potentially interfere with PCR amplification. Repeats containing vector
multiple copies in all organisms that had been examined at sequences introduced during genome sequencing were identified by a com-
the time. The availability of complete Plasmodium genome parison with the NCBI UniVec database (build 5.2; http://www.ncbi.nlm.nih
.gov/VecScreen/UniVec.html) (with WU-BLAST [blastn ver. 2.0; http://blast
sequences presents a great opportunity for improving the
.wustl.edu]) with an E-value cutoff of 1E⫺10. To ensure that targets were not
existing molecular diagnostic tools by identifying new tar- also present in the human genome, CRS were compared to human genome
gets for more sensitive and specific detection. The P. falcip- sequences (RefSeq, Primary Reference Assembly, build 37, version 1) with
arum genome was completed in 2002 (9), and P. vivax and P. BLAST (1) (version 2.2.22, blastn), with an E-value cutoff of 1E⫺10. Screens
knowlesi have since been sequenced (5, 26). Despite the were applied in parallel to all CRS. Any sequence failing a screen was
removed from further consideration. A total of 165 P. falciparum sequences
existence of genomic information for three of the five hu- and 331 P. vivax sequences passed all screens. All P. falciparum and P. vivax
man-infecting malaria parasites for many years, the majority CRS were compared (WU-BLAST) to all available Plasmodium sequence
of molecular diagnostic tools still rely on 18S rRNA. Sub- data, and the results were manually inspected to ensure species specificity. To
sequent examination of Plasmodium genome sequences has allow sufficient space for primer design and the evaluation of repeat family
conservation, CRS smaller than 300 bp were not considered further. CRS
revealed that the 18S rRNA target is present in only 4 to 8
were used to calculate the copy number of each repeat. Each screened repeat
divergent, nontandem copies, depending upon the species, was used to search (WU-BLAST) against the species’ genome from which it
in contrast to the case for other eukaryotic genomes that was derived. Repeat copies were required to hit to the CRS with an E value
have hundreds of tandem copies of rRNA gene clusters (18, of less than 1E⫺50 for P. vivax. The stringency for P. falciparum was relaxed
19). In addition, the few 18S rRNA sequences that are to 1E⫺10 because lower E-value requirements did not produce sufficient
candidates for screening. A minimum distance of 100 bp between copies was
present are not identical in sequence and are variably ex- required to remove potential amplification complications. Repeat families
pressed during the parasite life cycle (15). As PCR sensitiv- with at least 6 copies were considered for further testing, yielding totals of 21
ity is greatly influenced by the starting target molecule copy P. falciparum and 68 P. vivax candidates.
VOL. 49, 2011 NEW MALARIA DIAGNOSTIC TARGETS 2413

(MgCl2 concentrations from 2.0 mM to 4.0 mM were tested) (see below for
final conditions). Primers were further tested for species specificity using
laboratory cultures of P. falciparum (3D7) or DNA stocks of P. vivax (SV4),
P. malariae, P. ovale, and P. knowlesi.
Plasmodium parasites. P. falciparum strains 3D7, W2, V1-S, Dd2, HB3, D6,
and FCR3 were cultured in our laboratory. DNA stocks of P. vivax (Sal-1, SV4,
and NAM/CDC), P. ovale, P. malariae, and P. knowlesi and filter paper blood
spots of additional P. vivax strains (from Thailand, North Korea, Vietnam, India,
Miami [FL], New Guinea, South Vietnam, and Brazil) were all provided by John
Barnwell (CDC). DNA was isolated using commercially available QIAamp DNA
minikits (Qiagen, Valencia CA), following the manufacturer’s instructions.
Nested PCR. Nested PCR for malaria parasite detection (as described by
Singh et al. [32]) was used as the standard method for comparison.
Amplification of CRS targets by PCR. Amplification of CRS targets was
performed in a 25-␮l reaction mixture containing 1⫻ Taq buffer (contains 10 mM
Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2; New England BioLabs, Ipswich,
MA), 4 mM MgCl2, 200 ␮M each deoxynucleoside triphosphate (dNTP), 500 nM
each oligonucleotide primer, 1.25 units of Taq DNA polymerase (New England
BioLabs), and 1 ␮l of DNA template. Oligonucleotide primers for P. falciparum
candidate Pfr364 and P. vivax candidate Pvr47 are shown in Table 1. Separate
reactions were performed for P. falciparum and P. vivax with the following cycling
parameters: initial denaturation at 95°C for 2 min and then 35 cycles of 95°C for
30 s, 57°C (for P. falciparum) or 54°C (for P. vivax) for 30 s, and 72°C for 45 s,
followed by final extension at 72°C for 5 min. PCR products were visualized by
gel electrophoresis on a 2% agarose gel.
Serial dilutions of quantified parasite DNA, isolated from laboratory cul-
tures, were used to determine the detection limits (DNA concentrations
ranging from 10,000 parasites/␮l to 0.01 parasites/␮l were tested). Final
validation of targets was performed with P. falciparum and P. vivax clinical
samples from Tanzania (n ⫽ 91; median parasitemia, 3,200 parasites/␮l) and
Venezuela (n ⫽ 96; no parasitemia data are available), respectively, as well as
with additional geographically diverse strains for both targets (for Pfr364, P.
falciparum strains W2, V1-S, Dd2, HB3, D6, and FCR3; for Pvr47, P. vivax
isolates from Thailand, North Korea, Vietnam, India, Miami, New Guinea,
FIG. 1. Schematic of diagnostic target screening and development
pipeline. All genomic sequences for P. vivax and P. falciparum were South Vietnam, and Brazil).
downloaded from PlasmoDB. Data were mined for repeats using the Multiplex PCR. The multiplex PCR platform was optimized by gradient PCR
RepeatScout algorithm to construct consensus repeat sequences cycling to determine the annealing temperature, with additional adjustments to
(CRS) for each identified repeat family. CRS were then screened in primer concentrations (0.25 to 1.0 ␮M were tested) and master mix component
parallel for tandem repeats, similarity to human sequences, and vector concentrations (MgCl2 from 2.0 mM to 4.0 mM, dNTPs from 200 ␮M to 400 ␮M
sequences. Any CRS failing these screens were removed from further each, and Taq DNA polymerase from 1.25 units to 2.5 units were all tested).
consideration. CRS that were not species specific or less than 300 bp Multiplex PCR for detecting P. falciparum and P. vivax was performed in a 25-␮l
long were eliminated. Family copy numbers for the remaining candi- reaction mixture containing 1⫻ Taq buffer (New England BioLabs, Ipswich MA;
dates were determined via comparison of the CRS against the appro- contains 10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl2), 4 mM MgCl2, 400
priate genome data. Candidate repeat families containing 6 or more ␮M each dNTP, 1,000 nM each P. falciparum primer, 600 to 800 nM each P. vivax
copies separated by at least 100 bp were considered for further testing. primer, 2.5 units of Taq DNA polymerase (New England BioLabs, Ipswich, MA),
For additional information and clinical sample validation, see Materi- and 1 ␮l of DNA template. The alternate P. falciparum oligonucleotide primer
als and Methods. sequences (Table 1) were used in the multiplex assay. The P. vivax primers were
the same as used in the conventional PCR described above. The reaction was
carried out under the following cycling parameters: initial denaturation at 95°C
Target validation. Primers were designed to test six P. falciparum and seven for 2 min and then 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s,
P. vivax CRS families. Primers were designed manually to candidate targets followed by final extension at 72°C for 5 min. All possible combinations of
and screened for GC content, melting temperature, secondary structure, and 10-fold dilutions ranging from 10,000 parasites/␮l to 0.01 parasites/␮l for each
primer dimer-forming potential using Primer Explorer version 2.0 (http: species were tested. PCR products were visualized by gel electrophoresis on a 2%
//primerexplorer.jp/e/). Primer pairs were optimized using gradient PCR cy- agarose gel.
cling on Bio-Rad iCycler machines to determine the optimum annealing Sensitivity and specificity calculations. Sensitivity and specificity (95% confi-
temperature, with additional adjustments to primer concentration (concen- dence interval) were calculated using the nested 18S rRNA PCR as the gold
trations from 0.25 ␮M to 1.0 ␮M were tested) and master mix components standard for distinguishing a true positive from a false positive (Table 2).

TABLE 1. New target primer sequencesa

Sequence
Primer
P. falciparum Pfr364 P. vivax Pvr47

Forward 5⬘-CCATTTTACTCGCAATAACGCTGCAT 5⬘-CTGATTTTCCGCGTAACAATG

Reverse 5⬘-CTGAGTCGAATGAACTAGTCGCTAC 5⬘-CAAATGTAGCATAAAAATCYAAG
Alt-Forward 5⬘-CCGGAAATTCGGGTTTTAGAC
Alt-Reverse 5⬘-GCTTTGAAGTGCATGTGAATTGTGCAC
a
Primer sequences designed to targets Pfr364 and Pvr47. The alternate (Alt) primer pair for P. falciparum was used in multiplex reactions only. The P. vivax primer
set was the same for both single-species and multiplex PCRs.
2414 DEMAS ET AL. J. CLIN. MICROBIOL.

TABLE 2. Sensitivities and specificities of new PCR assays Detection of P. vivax and P. falciparum. Primers designed to
compared to standard nested 18S rRNA PCRa Pfr364 and Pvr47 (Table 1) specifically identified P. falciparum
Result with: and P. vivax, respectively. Other Plasmodium species, including
P. malariae, P. ovale, and P. knowlesi, were not amplified. No
18S rRNA New primers Sensitivity Specificity
Species amplification was observed using human nonmalaria DNA
nested (%) (%)
No. No. (data not shown). Using known quantities of laboratory-cul-
PCR (n) positive negative
tured parasites, we were able to consistently detect parasites in
P. falciparum Positive (91) 91 0 100 100 concentrations of as low as 10 to 0.1 parasites/␮l, compared to
Negative (9) 0 9 10 to 1 parasites/␮l detected with the standard method (Fig. 4
P. vivax Positive (96) 95 1 98.9 100 and Table 3). P. falciparum candidate Pfr364 detected between
Negative (13) 0 13 10 and 0.1 parasites/␮l of DNA (detected 0.1 parasites/␮l twice
a
and 10 parasites/␮l once). For each repeat target, single am-
The sensitivities and specificities of the new PCR assays were compared to
those of standard nested 18S rRNA PCR (32). For conventional PCR, sensitivity plified products were clearly defined on a 2% agarose gel
and specificity were calculated using 96 P. vivax samples from Venezuela and 91 stained with ethidium bromide.
P. falciparum samples from Tanzania. DNA from nonmalarious patients was Specificity and sensitivity. The targets were further vali-
included as a negative control.
dated in three ways. First, microscopically determined P. vivax
samples from Venezuela (n ⫽ 96) and P. falciparum samples
from Tanzania (n ⫽ 91) were used. In comparison to standard
RESULTS
nested 18S rRNA PCR, Pvr47 had 98.9% sensitivity and 100%
Repeat mining and screening of diagnostic candidates. A specificity, and Pfr364 had 100% sensitivity and 100% speci-
semiautomated bioinformatics pipeline was constructed for ge- ficity. Second, target amplification in 7 P. falciparum strains
nome repeat mining and in silico candidate screening (Fig. 1) and 10 P. vivax strains from around the world was assessed.
(see Materials and Methods). Six P. falciparum and seven P. The target was successfully amplified in each case (Fig. 5).
vivax putative targets were identified for validation. Over 50 Finally, PlasmoDB was queried to assess the number and dis-
primer pairs were designed to these targets and empirically tribution of single-nucleotide polymorphisms (SNPs) in the 41
tested in conventional PCR amplification assays and multiplex P. falciparum repeats using data reported previously (13, 22,
assays. Of these targets, the most effective were P. falciparum 39). These data represent information from 21 P. falciparum
candidate Pfr364 and P. vivax candidate Pvr47, as these targets strains. There are an average of 50 polymorphic sites along the
consistently performed with the greatest sensitivity and speci- ⬃1,500-nucleotide (nt) length of each of the Pfr364 repeats,
ficity. The functions of Pfr364 and Pvr47 are not known. Nei-
ther sequence is annotated or encodes protein. However,
regions of Pfr364 are expressed according to PlasmoDB. Full-
length sequence alignments and repeat coordinates can be
found in Fig. S1 and S2 and Table S1 in the supplemental
material.
Diagnostic targets: copy number and distribution. At least
one putative target from each species was found to significantly
improve existing diagnostic capabilities. Pfr364 exists in 41
copies, each of which is localized to the SB2 subtelomeric
repeat region found on most chromosome ends (Fig. 2). The
size of the SB2 region of P. falciparum chromosomes is variable
(1 to 3 kb, though it may contain up to 6 kb of additional
sequence) and is composed of different repeat types (9). Many
regions were found to contain two proximal copies of Pfr364,
and chromosome 6 contains three copies at its 3⬘ end (data not
shown). Multiple alignment reveals significant subfamily struc-
ture resulting in two related alignment groups, which we have
designated subfamilies 1 and 2 (Fig. 3A; see Fig. S2 and Table
S1 in the supplemental material). Interestingly, when multiple
copies of Pfr364 are found at chromosome ends, there is one
member of each subfamily present (Fig. 2).
Pvr47 is found in 14 copies (Fig. 3B; see Fig. S1 and Table S1
in the supplemental material). All members are located on
contigs that have not yet been assigned to chromosome scaf-
folds. The majority of these members map to small (⬍16-kb) FIG. 2. Spatial distribution of Pfr364 family members across the 14
subtelomeric contigs that could not be assembled onto chro- P. falciparum chromosomes. Tick marks indicate 200 kb of sequence.
mosomes due to their repetitive nature (5). Two of these family Pfr364 family members occur in two proximal copies at most chromo-
some ends. Black lines represent the outermost copies (subfamily 1),
members are located proximal to annotated vir genes, while a and gray lines represent the innermost copies (subfamily 2). Chromo-
third is located proximal to the subtelomeric transmembrane some 6 has three copies at its 3⬘ end (only two are shown). Circos 0.51
protein Pvstp1 (6). (http://mkweb.bcgsc.ca/circos/) was used to generate this map.
VOL. 49, 2011 NEW MALARIA DIAGNOSTIC TARGETS 2415

FIG. 3. Alignments of Pfr364 and Pvr47 family members with PCR primers. (A) Pfr364 with primers. Arrows represent locations of PCR
primers in context of the full alignment. The full alignment is 1,538 positions in length; here a partial alignment is shown. Vertical black lines
indicate where the sequence alignment has been truncated to enable viewing of all 4 primer locations. The alignment shows two subfamilies within
Pfr364. We have designated the upper 22 sequences subfamily 1 and the lower 19 sequences subfamily 2. Forward and reverse primer pairs used
for multiplex and conventional PCR are, respectively, the last two sequence pairs in the alignment. (B) Pvr47 with primers. Arrows represent
locations of PCR primers in context of the full alignment. The full alignment is 1,070 positions; here only positions 433 to 776 are shown. Vertical
lines indicate where the sequence alignment has been truncated for easier viewing. Forward and reverse primers are, respectively, the last two
sequences in the alignment.

for an average of 3% each. An average of 2 different nucleo- DISCUSSION

tides are observed at each polymorphic position (see Table S2
in the supplemental material). Here we show the value of applying bioinformatics methods
Multiplex assay. The multiplex PCR assay with combined and mining genomic data to answer biological questions that
Pvr47 and Pfr364 specifically detected P. vivax and P. falcipa- address practical needs. This approach can be applied to ad-
rum and correctly identified both single- and mixed-species ditional pathogens or used to improve existing molecular di-
infections. An alternative P. falciparum primer was used to agnostic tools (LAMP, real-time PCR, etc.). Increasing the
make the PCR products similar in size to increase efficiency sensitivity and specificity of molecular assays will facilitate
(see Materials and Methods) (Table 1). The limit of detection greater high-throughput detection of pathogens.
for the multiplex platform was determined using “mock mixed” Discovery of the exact locations of Pvr47 repeats will depend
infections of P. falciparum and P. vivax laboratory cultures. on the continued refinement of the P. vivax genome assembly
This method had a limit of detection of 10 parasites/␮l for each and improved annotation. The presence of some members
species (Fig. 6). P. falciparum DNA was also detected at 1.0 near genes known to be located in subtelomeric regions (see
parasites/␮l when P. vivax was present at the same concentra- above), combined with the known subtelomeric location of
tion (P. vivax was not detected). Clinical mixed P. falciparum-P. Pfr364, points to an interesting role in Plasmodium chromo-
vivax samples from Venezuela (n ⫽ 11) were detected with some end biology for use in diagnostic target development.
90.9% sensitivity and 100% specificity in comparison to the There has been no comprehensive, systematic study of the
standard nested PCR method, which was performed as sepa- genomic repeats of the genus Plasmodium. Our understanding
rate reactions for the different species. of the organization and content of subtelomeric regions is
2416 DEMAS ET AL. J. CLIN. MICROBIOL.

FIG. 5. Evaluation of Pfr364 and Pvr47 primers on geographically

diverse field isolates. (A) Pfr364 primers tested on various P. falcipa-
rum isolates. Lanes: 1, 3D7; 2, W2; 3, V1-S; 4, Dd2; 5, Hb3; 6, D6; 7,
FIG. 4. Limits of detection for conventional PCR assays. Primers FCR3. A 100-bp standard ladder (L) and a no-template control
to novel targets P. falciparum Pfr364 (A) and P. vivax Pvr47 (B) were (N) were included. (B) Pvr47 primers tested on various P. vivax field
used to amplify parasite DNA of the appropriate species. DNA was isolates. Lanes: 1, Thailand; 2, North Korea; 3,Vietnam; 4, India; 5,
quantified, and 10-fold serial dilutions from 10,000 parasites/␮l (lane NAM/CDC; 6, Miami; 7, New Guinea; 8, Sal-1; 9, South Vietnam; 10,
1) to 0.01 parasites/␮l (lane 7) were used to determine the limit of Brazil. The Pfr364 and Pvr47 primers clearly detect all of the tested
detection. A 100-bp standard ladder (L) and a no-template control isolates. Pfr364 primers detected an additional 91/91 (100%) P. falcip-
(NTC) were included. arum isolates from Tanzania, and Pvr47 primers detected an additional
95/96 (98.9%) P. vivax isolates from Venezuela (not shown).

largely restricted to what is known in P. falciparum, where it

has been shown that these regions contain genes responsible
for host immune evasion and antigenic variation (9). Given the targets are as robust to evolutionary change as the 18S rRNA
biological importance of these regions and the useful diagnos- target, despite the uncertainty about their biological roles.
tic targets that they contain, it is critical that we increase our Pfr364 and Pvr47 are not necessarily the most abundant
understanding of their repeat content and organization. repeats in these genomes. We tested only a few repetitive
While there is evidence for their location and distribution, sequences resulting from our data mining for their potential as
the biological functions of Pfr364 and Pvr47 are not yet estab- diagnostic targets. It is possible that more sensitive targets
lished. Combined with their repetitive, potentially nongenic exist. As we have noted above, there has been no comprehen-
nature, this necessitates a thorough evaluation of their robust-
ness as diagnostic targets. Sequences with no coding potential
often evolve more quickly than coding regions (17). However,
we show that these families are highly conserved (⬍3% varia-
tion at the nucleotide level in P. falciparum), which is indicative
of selection. Further, assays designed to the targets were able
to detect infections across as large a range of field isolates (7 P.
falciparum strains and 10 P. vivax strains) as the standard
nested 18S rRNA PCR. These observations suggest that these

TABLE 3. Detection limits of new diagnostic targets

Detection limit (parasites/␮l)a
Replicate
P. falciparum P. vivax

1 0.1 10
FIG. 6. Multiplex PCR. The multiplex method clearly identified
2 0.1 1
mock mixed P. falciparum and P. vivax infections (lane Pf/Pv). Single-
3 10 10
species infections (lanes Pf and Pv) were also detected. The P. falcip-
a
Calculated using 10-fold serial dilutions of P. falciparum and P. vivax DNAs arum band appears at 220 bp and the P. vivax band at 333 bp. A 100-bp
(see Fig. 4). standard ladder (L) and a no-template control (NTC) were used.
VOL. 49, 2011 NEW MALARIA DIAGNOSTIC TARGETS 2417

sive investigation of the genomic repeat content of these or- tified Pfr364 and Pvr47 targets are valuable tools to improve
ganisms, and our analysis is ongoing. and simplify molecular diagnostic methods for field use.
Amplification of the novel targets presented here was highly
sensitive and specific. Both assays have a detection limit 10- ACKNOWLEDGMENTS
fold lower than the historic standard and utilize a single, as We thank Jatan Patel and Zubin Mehta for their bioinformatics
opposed to nested, PCR. This is an important improvement, as assistance in screening putative target sequences.
single-round, unnested PCRs have fewer steps, decrease the This work was supported in part by a CDC-UGA seed grant (OPHR
chances of contamination or error, decrease the overall cost in no. 8212) awarded to J.C.K. and V.U. This study was supported in part
by resources and technical expertise from the University of Georgia
materials, and require less time to complete. The standard Research Computing Center, a partnership between the Office of the
nested protocol requires two separate reactions, and the am- Vice President for Research and the Office of the Chief Information
plified product of the first reaction must be transferred to a Officer. A.D. was supported by an EID Fellowship from the Associa-
second tube prior to the second reaction. Opening the tubes tion of Public Health Laboratories and the CDC. N.W.L. and A.D.
(after the EID Fellowship) were supported by the Atlanta Research
increases the risks of contamination and human error and also and Education Foundation, Atlanta, GA. J.D. was supported by NIH
increases the time and costs for necessary reagents and con- grant R01 AI068908 awarded to J.C.K.
sumables.
The targets produced clean products, clearly visible on an REFERENCES
agarose gel stained with ethidium bromide, at 716 bp and 333 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local alignment search tool. J. Mol. Biol. 215:403–410.
bp for P. falciparum and P. vivax, respectively. There were no 2. Barcus, M. J., et al. 2007. Demographic risk factors for severe and fatal vivax
nonspecific bands in clinical samples, including negative sam- and falciparum malaria among hospital admissions in northeastern Indone-
sian Papua. Am. J. Trop. Med. Hyg. 77:984–991.
ples, as was sometimes found with the standard PCR method 3. Benson, G. 1999. Tandem repeats finder: a program to analyze DNA se-
(data not shown). While DNA amplified from laboratory cul- quences. Nucleic Acids Res. 27:573–580.
tures using the standard nested PCR method showed clean 4. Bronzan, R. N., M. L. McMorrow, and S. P. Kachur. 2008. Diagnosis of
malaria: challenges for clinicians in endemic and non-endemic regions. Mol.
bands on the agarose gel, clinical samples often produced Diagn. Ther. 12:299–306.
nonspecific bands with sizes similar to those of the expected 5. Carlton, J. M., et al. 2008. Comparative genomics of the neglected human
malaria parasite Plasmodium vivax. Nature 455:757–763.
bands when the 18S rRNA gene-based method was used. This 6. del Portillo, H. A., et al. 2001. A superfamily of variant genes encoded in the
can be especially confusing when interpreting the results, and subtelomeric region of Plasmodium vivax. Nature 410:839–842.
additional time was required to fully separate the bands by 7. Erdman, L. K., and K. C. Kain. 2008. Molecular diagnostic and surveillance
tools for global malaria control. Travel Med. Infect. Dis. 6:82–99.
electrophoresis. The nonspecific bands appeared when P. fal- 8. Gamboa, D., et al. 2010. A large proportion of P. falciparum isolates in the
ciparum samples were tested to amplify field samples with the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria
rapid diagnostic tests. PLoS One 5:8.
P. vivax-specific primers (unpublished observation). Addition- 9. Gardner, M. J., et al. 2002. Genome sequence of the human malaria parasite
ally, sometimes several rounds of repetition of the standard Plasmodium falciparum. Nature 419:498–511.
method described by Singh et al. (32) were necessary to con- 10. Genton, B., et al. 2008. Plasmodium vivax and mixed infections are associated
with severe malaria in children: a prospective cohort study from Papua New
firm the results for the clinical samples tested. We found that Guinea. PLoS Med. 5:e127.
PCR amplification with the newly identified targets yielded 11. Han, E. T., et al. 2007. Detection of four Plasmodium species by genus- and
species-specific loop-mediated isothermal amplification for clinical diagno-
consistent clear results with no spurious bands among the sis. J. Clin. Microbiol. 45:2521–2528.
clinical samples tested in this study. 12. Harris, I., et al. 2010. A large proportion of asymptomatic Plasmodium
One-step multiplex reactions will offer a great improvement infections with low and sub-microscopic parasite densities in the low trans-
mission setting of Temotu Province, Solomon Islands: challenges for malaria
to existing Plasmodium diagnostics. Efficient, high-throughput diagnostics in an elimination setting. Malar. J. 9:254.
pathogen detection will decrease the time to results and ap- 13. Jeffares, D. C., et al. 2007. Genome variation and evolution of the malaria
parasite Plasmodium falciparum. Nat. Genet. 39:120–125. (Erratum, 39:567.)
propriate treatment. Mixed infections naturally occur in re- 14. Kochar, D. K., et al. 2009. Severe Plasmodium vivax malaria: a report on
gions where multiple parasite species are found, and these serial cases from Bikaner in northwestern India. Am. J. Trop. Med. Hyg.
present a challenge for diagnosis. To validate our multiplex 80:194–198.
15. Li, J., et al. 1997. Regulation and trafficking of three distinct 18 S ribosomal
method, we tested all possible combinations of 10-fold DNA RNAs during development of the malaria parasite. J. Mol. Biol. 269:203–
concentrations (from 10,000 parasites/␮l to 1 parasite/␮l) to 213.
cover all the range of naturally occurring mixed infections 16. Li, J., et al. 1995. Plasmodium: genus-conserved primers for species identi-
fication and quantitation. Exp. Parasitol. 81:182–190.
(data not shown). The limit of detection (10 parasites/␮l for P. 17. Li, W.-H., and D. Graur. 1991. Fundamentals of molecular evolution.
falciparum and P. vivax) compares favorably to those for other Sinauer Associates, Inc., Sunderland, MA.
18. Long, E. O., and I. B. Dawid. 1980. Repeated genes in eukaryotes. Annu.
multiplex methods. In mixed-species infections, one major spe- Rev. Biochem. 49:727–764.
cies will frequently dominate over another that is present in 19. Mercereau-Puijalon, O., J. C. Barale, and E. Bischoff. 2002. Three multigene
relatively low concentrations during PCR amplification (27, families in Plasmodium parasites: facts and questions. Int. J. Parasitol. 32:
1323–1344.
36). In contrast, the current method detects both major and 20. Mixson-Hayden, T., N. Lucchi, and V. Udhayakumar. 2010. Evaluation of
minor species of mixed infection, providing another advantage three PCR-based diagnostic assays for detecting mixed Plasmodium infec-
tion. BMC Res. Notes 3:88.
of using this method for diagnosis. 21. Moody, A. 2002. Rapid diagnostic tests for malaria parasites. Clin. Microbiol.
In conclusion, the findings from this study demonstrate that Rev. 15:66–78.
using bioinformatics to identify novel genetic targets for diag- 22. Mu, J. B., et al. 2010. Plasmodium falciparum genome-wide scans for positive
selection, recombination hot spots and resistance to antimalarial drugs. Nat.
nostic application is a valid approach. This methodology will be Genet. 42:268–U113.
extended to identify additional targets from other Plasmodium 23. Mueller, I., et al. 2009. Key gaps in the knowledge of Plasmodium vivax, a
neglected human malaria parasite. Lancet Infect. Dis. 9:555–566.
species for diagnostic assays when the genome sequences be- 24. Notomi, T., et al. 2000. Loop-mediated isothermal amplification of DNA.
come available. Our results demonstrate that the newly iden- Nucleic Acids Res. 28:E63.
2418 DEMAS ET AL. J. CLIN. MICROBIOL.

25. Padley, D., A. H. Moody, P. L. Chiodini, and J. Saldanha. 2003. Use of a 33. Singh, B., et al. 2004. A large focus of naturally acquired Plasmodium
rapid, single-round, multiplex PCR to detect malarial parasites and identify knowlesi infections in human beings. Lancet 363:1017–1024.
the species present. Ann. Trop. Med. Parasitol. 97:131–137. 34. Snounou, G., S. Viriyakosol, W. Jarra, S. Thaithong, and K. N. Brown. 1993.
26. Pain, A., et al. 2008. The genome of the simian and human malaria parasite Identification of the four human malaria parasite species in field samples by
Plasmodium knowlesi. Nature 455:799–803. the polymerase chain reaction and detection of a high prevalence of mixed
27. Polz, M. F., and C. M. Cavanaugh. 1998. Bias in template-to-product ratios infections. Mol. Biochem. Parasitol. 58:283–292.
in multitemplate PCR. Appl. Environ. Microbiol. 64:3724–3730. 35. Snounou, G., et al. 1993. High sensitivity of detection of human malaria
28. Price, A. L., N. C. Jones, and P. A. Pevzner. 2005. De novo identification of parasites by the use of nested polymerase chain reaction. Mol. Biochem.
repeat families in large genomes. Bioinformatics 21(Suppl. 1):i351–i358. Parasitol. 61:315–320.
29. Price, R. N., N. M. Douglas, and N. M. Anstey. 2009. New developments in
36. Suzuki, M. T., and S. J. Giovannoni. 1996. Bias caused by template annealing
Plasmodium vivax malaria: severe disease and the rise of chloroquine resis-
in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ.
tance. Curr. Opin. Infect. Dis. 22:430–435.
Microbiol. 62:625–630.
30. Rougemont, M., et al. 2004. Detection of four Plasmodium species in blood
from humans by 18S rRNA gene subunit-based and species-specific real-time 37. Taylor, S. M., et al. 2010. High-throughput pooling and real-time PCR-based
PCR asays. J. Clin. Microbiol. 42:5636–5643. strategy for malaria detection. J. Clin. Microbiol. 48:512–519.
31. Rubio, J. M., et al. 2002. Alternative polymerase chain reaction method to 38. Tjitra, E., et al. 2008. Multidrug-resistant Plasmodium vivax associated with
identify Plasmodium species in human blood samples: the semi-nested mul- severe and fatal malaria: a prospective study in Papua, Indonesia. PLoS Med.
tiplex malaria PCR (SnM-PCR). Trans. R. Soc. Trop. Med. Hyg. 96(Suppl. 5:e128.
1):S199–S204. 39. Volkman, S. K., et al. 2007. A genome-wide map of diversity in Plasmodium
32. Singh, B., et al. 1999. A genus- and species-specific nested polymerase chain falciparum. Nat. Genet. 39:113–119.
reaction malaria detection assay for epidemiologic studies. Am. J. Trop. 40. WHO. 2010. World malaria report 2008. World Health Organization, Ge-
Med. Hyg. 60:687–692. neva, Switzerland.