REAL-TIME PCR WITH HIGH-RESOLUTION MELTING ANALYSIS FOR

DIAGNOSIS OF LYMPHATIC FILARIASIS

Sirichit Wongkamchai1, Benjaporn Mayoon2, Darawan Wanachiwanawin1, Suporn Foongladda2,
JJ Boitano1, Hathai Nochote1, Sumat Loymak3.
Department of Parasitology, 2Department of Microbiology, Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, 3Filaria Project, Phikulthong Royal Development Study Center,
Narathiwat Province, Thailand.

Abstract
Lymphatic filariasis (LF), or elephantiasis, is caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi,
and B. timori. Over a billion people are at risk of contracting the disease, and about 40 million people suffer from
severe disfigurement and disability as a result. Diagnosis is an essential element in the management of the disease,
both at the individual patient care level and at the level of disease control in endemic populations. The success
of LF elimination programs has lowered the levels of microfilariae in blood samples, which in turn has reduced
the sensitivity of the conventional microfilariae detection method. In this study, we used real-time PCR with highresolution melting analysis (HRM) to detect and identify B. malayi and W. bancrofti in human blood samples.
The assay can be performed using a single pair of primers targeting the mitochondrial partial 12S rRNA genes of
these filarial parasites. Amplification and detection steps can be conducted in the same reaction, and no post-PCR
processing is required. After assessing 30 blood samples, the HRM assay exhibited a specific melting temperature
for each species, as well as high sensitivity and specificity compared with the microfilaria-detection method. Thus,
the assay could be helpful for the diagnosis of LF.
Keywords:

Brugia malayi, Wuchereria bancrofti, real-time PCR, high-resolution melting, lymphatic filariasis

INTRODUCTION
Lymphatic filariasis (LF), or elephantiasis,
is caused by three species of thread-like filarial
nematodes: Wuchereria bancrofti, Brugia
malayi, and Brugia timori. Of these, 90% of
all infections result from W. bancrofti, with
10% attributed to Brugia malayi and Brugia
timori (Ravindran et al., 2003). The disease
is transmitted by mosquito vectors; when
an infected mosquito bites, the infectivestage larvae (L3) migrate into the lymphatic
system; on reaching sexual maturity after 6
to 12 months, the adult female worms release
Correspondence: Sirichit Wongkamchai
Department of Parasitology, Faculty of
Medicine Siriraj Hospital, Mahidol University,
Bangkok, 10700, Thailand
Tel.: 66(0) 24196468
E- mail: sirichit.won@mahidol.ac.th
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millions of microfilariae into the bloodstream

(Garcia, 2007). The life cycle is completed
when these microfilariae are ingested by new
mosquito vectors (Semnani and Nutman,
2004).
The WHO estimates that over a billion
people in more than 80 countries are at risk of
contracting LF and over 120 million people
have already been infected with the disease,
with about 40 million people suffering from
severe disfigurement and disability (WHO,
1992).
Although it is easy to recognize classical
obstructive filariasis, many infected individuals
do not show signs or symptoms. Laboratory
confirmation of the infection is necessary for
the management of the disease, both at the
level of individual patient care and for disease
control in endemic populations. Thick blood
smear staining is the conventional method for
demonstrating and identifying microfilariae
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JITMM2013 PROCEEDINGS

circulating in blood. This method confers

diagnostic specificity, but low sensitivity in
people with minimal microfilarial counts. The
method is also unable to diagnose those who
are not microfilaremic (Weil et al., 1997).
In 1997, the World Health Organization
(WHO) established a global programme to
eliminate lymphatic filariasis (GPELF), which
aims to interrupt the transmission of LF through
mass drug administration. Mass drug treatment
reduced the prevalence of LF infection to levels
at which it is assumed transmission can no
longer be sustained (Molyneux et al., 2002).
In an address to the 7th GAELF mapping the
worldwide distribution of LF, Engels indicated
that 538.6 million people were treated in 20102011 (Engels et al., 2012). The WHO databank
of PCT reported that, as of 2011, 95.9% of
the endemic populations were treated with
diethylcarbamazine and/or albendazole (WHO,
2013).
The success of GPELF has decreased
the prevalence of LF and lowered levels of
microfilariae in blood samples, which in turn
has reduced the sensitivity of the microfilariadetection method. Furthermore, in places
outside endemic areas, filarial infections
are sporadically diagnosed in refugees and
immigrant workers. Long-term residents in
endemic areas, such as the armed services,
students, and volunteers, are also intermittently
diagnosed with the disease. Diagnosis of
filarial infections among these individuals
requires both strong clinical suspicion and
expert training in infrequently practiced
parasitological methods (Fink et al., 2011).
Due to these limitations, DNA-based methods
may be more efficient for the diagnosis of LF
infection.
In the present study, we applied a real-time
PCR with high-resolution melting analysis
(HRM) targeting the mitochondrial partial 12S
rRNA gene, which is highly conserved and
contains genus- and species-specific sequence
variations to detect and identify B. malayi and
W. bancrofti in human blood samples.
24

MATERIALS AND METHODS

Ethical approval
The study protocol was approved by
the Human Research Protection Unit of the
Faculty of Medicine Siriraj Hospital, Mahidol
University, based on the Ethics of Human
Experimentation of the National Research
Council of Thailand (Certificate of Approval
number Si285/2013).
Source of study
Positive and negative controls
Microfilariae of Brugia malayi and
Wuchereria bancrofti were used as positive
controls in the present study. Blood samples
obtained from healthy human subjects served
as negative controls.
Study samples
Microfilaria-positive blood samples were
obtained from 10 patients infected with
B. malayi and 5 patients infected with W.
bancrofti, and were used as study samples.
Fifteen blood samples obtained from malariainfected and healthy persons were also used to
study the specificity of the assay.
Extraction of DNA from whole blood
200L of blood from capillary tubes were
used for DNA extraction. A High Pure PCR
Template Preparation Kit (Roche Germany)
was used to extract DNA, while DNA
concentration was measured by Nanodrop
(Thermo Fisher Scientific) according to the
manufacturers instructions. The DNA was
used for the HRM real-time PCR.
Primers
Previously designed primers targeting the
mitochondrial partial 12S rRNA gene, which
is highly conserved and contains genus-and
species-specific sequence variations, were used
in this study (Wongkmachai et al., 2013). It was
found that the primer sequence also recognized
a 111-bp region of the mitochondrial partial
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JITMM2013 PROCEEDINGS

12S rRNA gene of W. bancrofti. A BLAST

search (www.ncbi.nlm.nih.gov/BLAST/)
was performed to check the specificity of
the primers to their respective DNA targets.
Figure 1 shows the primers that are based on
alignments of the mitochondrial partial 12s
rRNA genes of B. malayi (GenBank accession
number AJ544843; positions 177-287) and
W. bancrofti (GenBank accession numbers
NC_016186.1; positions 7619-7729).
Sensitivity and reproducibility of the realtime PCR with HRM analysis
The detection limit of the assay was
established. The lowest levels of B. malayi
and W. bancrofti were determined in 10-fold
serial dilutions of genomic DNA. To verify the
reproducibility of the assay, DNA from each
species was tested 5 times within the same run
(intra-assay variation) and on 3 different runs
on different days (inter-assay variation). Mean,
standard deviation (SD), and the coefficient of
variance (%CV) were calculated.
Real-time PCR with HRM analysis for
diagnosis of lymphatic filarial infection in
blood samples
A real-time PCR with HRM analysis was
performed in a single run on a LightCycler
LC480 instrument (Roche Diagnostics,
Germany). A 10L reactive mixture was
generated, which consisted of (a) 3L of
DNA, (b) 0.25pmol of the forward and reward

primers, and (c) 5L of the LightCycler 480

High-Resolution Melting Master mixture
containing Reso Light dye and 3mM MgCl2, in
PCR grade water. DNA extracted from normal
blood samples was used as the negative control.
Positive controls for B. malayi and W. bancrofti
were performed in each run. The reaction
conditions included an activation step at 95oC
for 5min, followed by a 40-step amplification
of 10s at 95oC, 10s at 58oC, and 10s at 72oC.
Subsequently, the products were heated to
95.8oC for 1 min and then cooled to 40oC
for 1 min. HRM was performed from 65oC
to 95oC, rising at 1oC/s with 25 acquisitions
per degree. The final cooling step was 40oC
for 10s. Melting curves were generated,
normalized, temperature-shifted, and converted
to difference plots by LightCycler 480 genescanning software (Roche Applied Science,
Germany).
For the studys blood samples, the DNA
from 15 microfilaremic human blood samples
as well as 15 human blood samples obtained
from patients infected with malaria (P. vivax
and P. falciparum), and healthy subjects, were
amplified and the resulting amplicons were
analyzed.
RESULTS
Compared with thick blood-smear staining,
the sensitivity, specificity, positive predictive
value (PPV), and negative predictive value

Fig. 1- Alignments of the mitochondrial partial 12s rRNA genes of B. malayi (GenBank accession number AJ544843;
positions 177-287) and W. bancrofti (GenBank accession numbers NC_016186.1; positions 7619-7729). Dots
indicate identity; dashes indicate deletion from the above consensus sequence; and gray areas indicate primers.

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JITMM2013 PROCEEDINGS

(NPV) were all 100%.

For intra-assay variation, Tms of B. malayi
and W. bancrofti revealed the mean+SD of
76.26+0.01 and 77.35+0.03oC with %CV at
0.02% and 0.18% respectively. The inter-assay
variation, Tms of B. malayi and W. bancrofti
showed the mean+SD at 76.23+0.01 and
77.28+0.19oC, with %CV at 0.19% and 0.21%,
respectively.

Figure 1 presents alignments of the

mitochondrial partial 12s rRNA genes of
B. malayi (GenBank accession number
AJ544843; positions 177-287) and W. bancrofti
(GenBank accession numbers NC_016186.1).
Figure 2 shows representative melting peaks
(Tms), normalized difference curves, and the
normalized and temperature-shifted difference
plot of the amplified product of the study

Fig. 2- Melting peaks (a, b), normalized difference curves (c, d), and the normalized and temperature-shifted difference
plot (e, f) of the amplified product of W. bancrofti and B. malayi, obtained by LightCycler 480 gene-scanning
software; 7-13 represent study blood samples and control samples (Bm, B. malayi and Wb, W. bancrofti).

26

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Table 1 Results of the HRM assay in human blood samples

Blood sample

Number

Amplification
curve

B. malayi mf positive

10

W. bancrofti mf positive

HRM results
Tm range
(C)

Interpretation

Positive

76.32 76.41

B. malayi

Positive

76.73 77.57

W. bancrofti

Malaria positive

Negative

Negative

Normal

10

Negative

Negative

Total

30

Mf = microfilariae, Tm = melting temperature

samples, obtained by LightCycler 480 genescanning software.

The Tm samples identified as B. malayi
showed mean+SD as 76.32+0.06, range as
76.24-76.41, and median as 76.34. The Tm of
the samples identified as W. bancrofti indicated
the mean+SD as 77.17+0.41, range as 76.7377.57, and median as 77.37. Table 1 shows the
results of the real-time PCR with HRM analysis
of all study samples.
DISCUSSION
LF diagnosis usually depends on clinical
features, history of exposure in endemic
areas, and laboratory findings. Diagnostic
tests performed in the laboratory include
microfilarial detection in peripheral blood and
serological detection (Mondal, 2012).
The detection of microfilariae in night
blood samples by thick blood smear staining
procedure, Knotts concentration procedure,
or membrane filtration technique through
polycarbonate filters, have been used for a
long time. Nevertheless, these traditional
parasitological methods have low sensitivity
and are unable to identify the disease in
non-microfilaremic individuals (Chanteau et
al, 1991). The current success of the global
program to eliminate LF has decreased
microfilarial levels, in turn decreasing
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the sensitivity of microfilarial detection.

Moreover, the migration of workers from
LF-endemic areas is increasing (Fink et al.,
2011), potentially carrying the parasites to
non-endemic areas (Poole and Williams, 1990;
Yen and Mak, 1982).
Recently, the nucleic acid detection
technique by polymerase chain reaction (PCR)
procedure has gained popularity. PCR can
demonstrate the presence of parasitic DNA
and is now the most sensitive technique in use
(Lizotte et al., 1994; Bockarie et al., 2000;
Kluber et al., 2001; Fischer et al., 2002;
Goodman et al., 2003; Helmy et al., 2004;
Rao et al., 2006). Both conventional PCR and
real-time PCR assays have been developed
for the detection and identification of many
filarial species. In LF, the PCR-based assay
offers a direct indication of active infection
(Rahmah et al., 2010). These assays have
shown great promise for high-level sensitivity
and specificity (Fink et al., 2011).
The present study used a real-time PCR
with HRM analysis to detect and identify two
LF infections in blood samples: W. bancrofti
and B. malayi. This PCR assay has been found
to have equal or higher sensitivity compared
with conventional parasitological methods.
This assay also demonstrated high specificity,
since no false positives were detected in any
blood sample obtained from malaria-infected
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JITMM2013 PROCEEDINGS

patients or healthy subjects.

Compared with conventional PCR, a realtime PCR with HRM has distinct advantages;
the amplification and detection steps can
be conducted in the same reaction tube or
well, and no post-PCR processing is required
(Dobrowolski et al., 2009; Nettuwakul et al.,
2010).
A real-time fluorescence resonance energy
transfer (FRET) PCR combined with a
melting curve analysis has been developed
for differentiating B. malayi and B. pahangi
DNA in blood samples. The FRET PCR used
one set of primers and fluorophore-labeled
hybridization probes (Thanchomnang et al.,
2010). The difference from FRET is that no
probe is required in a real-time PCR with
HRM analysis. The master mix used in the
HRM analysis contains a DNA-binding dye
that stains target sequences and results in sharp
melting signals that are analyzed using the
gene scanning software in the HRM instrument
(Dobrowolski et al., 2009). This means that
both parasites can be detected and identified
using a single pair of primers.
Compared with conventional parasitological
methods, the PCR assay had equal or a higher
sensitivity. Using a molecular technique
reveals a higher prevalence of filarial infections
compared with the levels detected from a
thick blood smear (Albrechtova et al., 2011;
Wongkamchai et al., 2014).
In conclusion, the sensitivity and specificity
of this assay make it a potentially useful tool
for the efficient diagnosis of LF in blood
samples. The method also uses a smaller
amount of blood, which could be collected
from a finger prick. Since no refrigeration is
required, samples may be transported by mail
from endemic areas to a laboratory where the
PCR assay may be performed. Furthermore,
the assay may be utilized for the diagnosis of
LF outside disease-endemic areas, where the
equipment and practical skills required for the
PCR procedure are available. The PCR does
not require specialized knowledge of parasite
28

morphology or classification, and can be run

by laboratory personnel with generalized
training (Fink et al., 2011). In addition to
the sensitivity and specificity factors, costeffectiveness is another essential component
of any diagnostic technique. The HRM realtime PCR procedure is cost-effective, since
it requires no expensive probe, and a large
number of samples can be identified in a single
run.
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