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Polymerase chain reaction-based detection of

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Article in Medical Microbiology and Immunology · March 2003

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Med Microbiol Immunol (2003) 192: 3–7
DOI 10.1007/s00430-002-0152-z
D E V E L O P M E N T S I N F I L AR IA SI S RE SE A RC H
Peter Fischer Æ Daniel Boakye Æ Joseph Hamburger
Polymerase chain reaction-based detection of lymphatic filariasis
Received: 22 July 2002 / Accepted: 23 August 2002 / Published online: 19 October 2002
Ó Springer-Verlag 2002
Abstract PCR-based diagnostic assays are promising
tools for the monitoring and evaluation of the Global
Programme for Elimination of Lymphatic Filariasis.
Sensitive and specific assays have been described for the
detection of Wuchereria bancrofti, Brugia malayi, or
Brugia timori infection in blood, sputum, and vectors.
These techniques can be most cost-effective when em-
ployed for pool screening, which is important in the later
stages of control programs when infection rates of hu-
mans and vectors are low, and large numbers of samples
must be examined.
Keywords Filariasis Æ Diagnosis Æ Blood Æ
Sputum Æ Mosquitoes
Introduction
To establish sensitive and specific PCR-based diagnos-
tics for lymphatic filariasis different target DNA repeats
have been identified for the filarial parasitesWuchereria
bancrofti and Brugia species. In Brugia a tandemly re-
peated sequence of about 320 bp (Figs. 1, 2) designated
HhaI repeat can be found in 30,000 copies (10% of the
genome) [20]. Similar highly repeated sequences appear
P. Fischer (&)
Bernhard Nocht Institute for Tropical Medicine,
Bernhard-Nocht-Strasse 74,
20359 Hamburg, Germany
E-mail: Pfischer@bni.uni-hamburg.de
Tel.: +49-40-42818486
Fax: +49-40-42818400
D. Boakye
Noguchi Memorial Institute for Medical Research,
University of Ghana,
Legon, Accra, Ghana
J. Hamburger
Kuvin Center for the Study of Infectious and
Tropical Diseases,
Hebrew University of Jerusalem, Israel
to be absent in W. bancrofti, but several moderately
repeated sequences have been identified [8, 19, 26, 28, 30,
32]. Based on one of these sequences termed the SspI
repeat, the oligonucleotide primers NV-1 and NV-2 have
been developed and used for most PCR diagnostic
studies of W. bancrofti. Using these primers a PCR
product of 188 bp is obtained (Fig. 3). As shown below,
the SspI repeat is a part of a longer dispersed repeat
(LDR1), a homologue of which may also be present in
Brugia parasites. In addition to these PCR targets, DNA
sequences encoding for rRNA or spacer regions between
rRNA genes can be used for PCR amplification of fi-
larial DNA. These target sequences are especially of
value when information about more highly repeated
sequences is not available [10, 21].
Although selection of target sequences is an impor-
tant step in the establishment of PCR assays, DNA
extraction, optimization of PCR conditions and cy-
cling, and convenient and sensitive detection of PCR
products are critical. Assays must be adapted depend-
ing on the sample from which filarial DNA should be
amplified.
Detection of parasite DNA in blood samples
The availability of a very sensitive and convenient card
test that detects W. bancrofti circulating antigen [29]
leaves detection of W. bancrofti infection in humans by
PCR with only a limited field applicability. However, the
test for circulating adult worm antigen remains positive
for some time although microfilaria densities have
dropped after treatment while PCR is only positive if
microfilariae or free DNA derived from them is present
in the blood. Our observations show that detectable free
DNA comes from dying microfilariae and is only stable
for a few days in human blood. Some studies require
information on the presence of microfilariae, and PCR
on blood samples can be a very sensitive alternative to
the conventional detection of microfilariae using par-
asitological methods. In contrast to W. bancrofti, no
4
Fig. 1 Detection of the HhaI repeat in B. malayi and in B. timori
by PCR. Lanes 1–4 Blood spots containing one microfilaria of B.
malayi; lanes 5–8 blood spots containing one microfilaria of B.
timori; lanes 9–12 negative control blood spots. A Detection of
biotinylated PCR products using a digoxygeninated DNA probe
and DNA Detection Test Strips; T test line; C control line; B
agarose gel; M molecular weight marker
sensitive and specific antigen test is available for the
detection of B. malayi and B. timori infection in humans.
Therefore PCR on blood samples (and in the future also
on sputum samples) is especially helpful for the detec-
tion of brugian filariasis.
Efficient DNA extraction is crucial for any PCR as-
say. A simple and inexpensive method for DNA prep-
aration is the lysis of red blood cells and the subsequent
digestion of white blood cells and microfilariae using
proteinase K [30]. This method has been used to prepare
DNA from infected individuals with low microfilaria
densities but with free parasite DNA and DNA in day
blood of microfilaremic persons since free DNA tends to
adhere to cells [12]. For convenient sample collection,
preservation, and subsequent DNA extraction small
amounts of venous or capillary blood can be collected
on 3MM Whatman filter paper, dried, and stored at
ambient temperature. DNA is then extracted by a simple
boiling method using Chelex 100 resin to bind PCR
inhibitors [16].
DNA of nocturnally periodic B. malayi can be de-
tected by PCR–enzyme-linked immunosorbent assay in
200 ll of night or day blood samples with a higher or at
least an equal sensitivity compared to the filtration of
1 ml night blood [12]. The PCR assay established for the
detection of B. malayi is also used to detect microfilariae
of B. timori since these two species appear to have an
identical HhaI repeat [31]. It was possible to detect a
single microfilaria of B. malayi or of B. timori with the
same assay (Fig. 1). An ongoing study in Indonesia
shows that the HhaI repeat of B. timori is a suitable
target for its PCR-based detection in human and vector
samples (P. Fischer, T. Supali, unpublished results).
A PCR pool-screening approach would be needed to
determine the presence of microfilariae within a com-
munity. In a pilot study we collected finger-prick blood
on filter paper and pooled four blood spots of 15 ll from
four noninfected individuals with one 15-ll night blood
spot of an infected person with low microfilaria density
Fig. 2 Detection of the HhaI repeat of B. malayi in pools of blood
spots by PCR. Lane 1 One 15 ll blood spot from a person with 1.2
microfilariae (mf) per 15 ll (80 mf/ml) and four negative blood
spots; lane 2 one 15 ll blood spot of a person with 0.8 mf per 15 ll
(53 mf/ml) and four negative blood spots; lane 3 one 15 ll blood
spot from a person with 0.6 mf per 15 ll (40 mf/ml) and four
negative blood spots; lanes 4–5 pools of five negative control blood
spots. A Detection of biotinylated PCR products using a
digoxygeninated DNA probe and DNA Detection Test Strips; T
test line; C control line; B agarose gel; M molecular weight marker
(40–80 microfilariae/ml). Two of three pools were posi-
tive by PCR, and it can be assumed that the negative
pool contained no microfilaria (Fig. 2). Although in
larger amounts of blood free parasite DNA can be de-
tected by PCR [12], free DNA can be rarely detected in
small blood spots (P. Fischer, T. Supali, unpublished
results). Other studies show that free DNA can be de-
tected only using a nested PCR approach [7], but this
bears a high risk of contamination and may limit its
application in many laboratories [5].
Detection of parasite DNA in sputum
There is a need to establish PCR assays based on human
material that can be collected noninvasively such as
urine or sputum. Lucena et al. [18] reported the detec-
tion ofW. bancrofti DNA in urine by PCR. However, it
is still not known whether this method is sensitive and
reliable enough for field application. Sputum PCR for
diagnosis of lymphatic filariasis is at this time still in its
stage of development and validation but is a promising
new tool.
Sputum PCR aims at adding further logistic advan-
tages to methods that are based on daytime sample
collection, such as antigen tests [29], and PCR assays for
testing daytime blood [12]. All blood-based diagnostic
tests share the problematic aspects of blood collection.
By comparison, the collection of sputum is noninvasive,
widely acceptable, can be carried out by village workers
with minimal training, enables storage and shipment of
samples at ambient temperature, can be performed over
a relatively long period of time for maximal represen-
tation of the target population, and is also relatively
inexpensive.
The putative presence of DNA of lymphatic filariae
in sputum derives support from a number of consider-
ations. (a) These largely nocturnally periodic parasites

Fig. 3 PCR pool screening to detect one W. bancrofti L3 in
different pools of adult A. gambiae s.s. heads using the dynabead
purification system and the SspI repeat as target. Agarose gel; M
molecular weight marker; lanes 1, 2 100 mosquitoes; lanes 3–4 75
mosquitoes; lanes 5, 6 50 mosquitoes; lanes 7, 8 25 mosquitoes; lane
9 10 mosquitoes; lane 10 positive control; lane 11 negative control
reside during the day in the microvasculature of the
lungs, readily available for clearance. Perhaps nematode
larval migration from the pulmonary blood vessels to
alveoli (as with ascarids, hookworms, etc.) is clearance
taken several steps further in evolution along the same
anatomical route. (b) The size of microfilariae (250–
300 lm in length) is not conducive to simple clearance
by phagocytosis, thus clearance of microfilariae through
the lungs is a logical alternative. (c) Tropical pulmonary
eosinophilia is a hypersensitivity response to microfila-
riae undergoing immune-clearance in the lungs
(reviewed in [24]). Expectoration of microfilarial constit-
uents is not unlikely under microfilarial clearance.
The ability to detectW. bancrofti DNA by sputum
PCR was initially demonstrated by testing a few diur-
nally collected sputum samples from patients in the
North Coast Province, Kenya [1]. A more extended
study then followed by testing sputum samples from
Kenyan patients exhibiting parasitological and/or clini-
cal evidence of lymphatic filariasis and from endemic
normals [2]. Collection of sputum in 0.2 M EDTA in-
hibits bacterial growth and enables storage at ambient
temperature for a several weeks [17]. A very simple al-
kaline DNA extraction that does not involve enzymes
and separation matrices was adapted for sputum PCR.
PCR primers employed in this study were derived from a
long dispersed repeat (LDR1) in the W. bancrofti ge-
nome. LDR1 was later shown to be a region of attach-
ment to nuclear scaffold/matrix proteins (S/MAR), the
first one in parasites (I. Abbasi, R. Ramzy, S.A.
Williams, J. Hamburger, submitted). S/MARs are highly
represented (20,000 copies or more per haploid genome)
in the genome of eukaryotes (reviewed in [4]), and such
high representation offers high detection sensitivity to
PCR assays employing corresponding primers. This is a
new approach for seeking suitable primers for identify-
ing eukaryotic parasites. The primers so far employed
with similar results, are AccI primers amplifying a 254-
bp-long segment of LDR1, and the SspI primers (NV-1,
NV-2) amplifying a 188-bp segment [2]. Of the total 34
sputum samples collected from patients with proven
infection, 32 (94%) were PCR positive, but those with
symptoms were 100% PCR positive suggesting that in
symptomatic patients microfilaria clearance is more
pronounced. Testing pools of sputum samples by PCR
5
(1 sample from an infected individual plus 14 samples
from uninfected ones) has also been carried out [2].
Standardization of sputum collection and large-scale
validation of sputum PCR are now in progress in
Kenya. Development of sputum PCR for diagnosis of
brugian filariasis is of particular importance but still
requires identification of suitable PCR primers. The
HhaI primers, although suitable for blood-PCR [12, 16,
17], are not as efficient for sputum-PCR. Since LDR1-
based primers successfully amplified Brugia DNA
(P. Fischer, T. Supali, I. Abbasi, J. Hamburger,
unpublished results) it can be tentatively assumed that
LDR1 homologue is present in Brugia DNA. Its iden-
tification may enable the design of Brugia-specific
primers for diagnosis of brugian filariasis by sputum-
PCR. This work is currently in progress.
Detection of parasite DNA in vectors
Detection of W. bancrofti and Brugia species in their
respective vectors has been an essential component in
determining areas at risk of infection, the transmission
potential of vectors, and also a direct indication that
transmission is occurring. Traditionally this has been
done by dissecting the mosquito vectors and examining
them under the microscope to morphologically identify
the parasites. This method is time consuming, labor in-
tensive, and prone to observer bias, particularly so when
infection levels in the vector populations are very low.
The development of an efficient, rapid, sensitive, specific,
and cost-effective tool to replace the classical dissection
method is therefore necessary to monitor and evaluate
intervention programs. The development of a PCR-
based pool-screening method paves the way for the
development of such a diagnostic tool.
A PCR assay amplifying 380- and 650-bp fragments
ofW. bancrofti DNA was initially developed for identi-
fication of infected mosquitoes [8]. This method yielded
low sensitivity due to PCR inhibitors from mosquito
material and lacked in test convenience since PCR
products were detected by Southern blot hybridization
[8]. The protocol was used to detect parasites in indi-
vidual mosquitoes and would be for monitoring pur-
poses not cost effective. Chanteau et al. [6] demonstrated
the possibility of using the PCR method to detect W.
bancrofti DNA in pools of mosquitoes and employed the
SspI repeat derived primers (NV-1 and NV-2) for im-
provement in sensitivity. Detection was carried out in
pools of 50 Aedes polynesiensis heads, and three DNA
extraction protocols were compared for this purpose [6].
The best results were obtained when a boiling and
freezing step was included. All recent studies except for
one [28] have also used these primers to detect W. ban-
crofti in mosquitoes.
Further improvement in test sensitivity by Nicolas
et al. [23] enabled the detection of a single mosquito
infected with one or two microfilariae of W. bancrofti
among 20–50 mosquitoes or one L3 in 50–100
6
A. polynesiensis. The PCR product was detected by a
characteristic band on an ethidium bromide stained
agarose gel and other more sensitive detection methods
for PCR products may even increase this sensitvity.
However, the assay was as sensitive as the dissection of
mosquitoes infected with W. bancrofti. The PCR method
was further evaluated by Ramzy and coworkers [14, 27]
on field-collected Culex pipiens from Egypt (see [14]). It
has also been evaluated on C. quinquefasciatus [13] and
on Anopheles punctulatus [3]. Recently Farid et al. [9]
have reported the potential for using the pool screening
in estimating W. bancrofti infection in pools of C. pipiens
from two villages with different prevalence rates of hu-
man filariasis. A drawback to the technique has been the
inconsistency sometimes observed, leading to false neg-
atives presumably due to the presence of PCR inhibitors.
Coamplification of parasite DNA together with an in-
ternal standard has been shown to overcome this
drawback [3, 11, 22].
Currently the PCR pool-screening method has been
developed to detect one infective W. bancrofti larva in a
pool of 13–50 C. pipiens, C. quinquefasciatus, A. polyne-
siensis, and A. punctulatus. No study has yet been re-
ported for detection in Anopheles gambiae s.l., an
important vector of W. bancrofti in Africa. For the PCR
detection to be cost effective there is the need to increase
the pool size and to improve the DNA extraction method.
One way of improving the extraction to obtain the
parasite DNA of interest is to use magnetic bead capture
system. We have used this procedure described below to
increase the pool size to 75–100 mosquitoes (Fig. 3).
The DNA was extracted according to the protocol of
Zimmerman et al. [33] and then purified using an equal
volume of 2.5 lmol labeled NV-1 capture primer and
Dynabead binding buffer (Dynal MPC -S, Oslo, Nor-
way) according to the instructions of the manufacturer.
Following denaturation, capture primer annealing, and
washing of the Dynabeads the DNA solution was in-
cubated overnight with the magnetic Dynabead parti-
cles. After several washing steps on the Dynal magnetic
particle concentrator the target DNA was separated
from the beads and removed into a new tube. Of the
supernatant 2 ll was used in each PCR.
The Dynabead purification method has recently been
used to detect W. bancrofti in members of the A. gam-
Table 1 Comparison of methods for the detection of PCR products in the diagnoses of lymphatic filariasis
Method Specificity Sensitivity Duration
Agarose gel Size specific >10 ng 2h
electrophoresis
Southern blot Sequence Approx. 1 ng 1 day
specific
Enzyme-linked Sequence 100 pg–5 ng 5h
immunosorbent specific
assay
DNA Detection Sequence Approx. 5 ng 30 min
Test Strips specific
biae s.s. from areas in Ghana where mass treatment with
ivermectin/albendazole is planned. Infection rates will be
estimated using the algorithm of Katholi et al. [15], and
the results will be compared with those obtained from
the classical dissection. The PCR has been shown to be
effective for detectingW. bancrofti in various mosquito
vector species and could be used to monitor the outcome
of intervention measures. In addition, no differences in
DNA preparation of mosquitoes infected with W. ban-
crofti or with Brugia species have been reported. Using
the HhaI repeat as target for PCR B. malayi and
B. timori can be sensitively detected in vectors and dif-
ferentiated from animal parasites such as B. pahangi
(T. Supali, H. Wibowo, P. Fischer, unpublished results).
Applications of PCR assays
PCR-based assays can be employed for individual di-
agnosis of lymphatic filariasis, but more importantly for
the identification of endemic areas and for the moni-
toring of intervention programs in humans and vectors
[25]. For the latter purposes examination of pooled
samples is most efficient. In areas with high microfilaria
densities in humans and high infection rates of vectors
parasitological methods are superior to PCR-based
techniques, but the opposite is true for areas with low
endemicity or advanced control programs. PCR meth-
ods have been improved over the last decade and are
now more suitable to be employed in laboratories of
endemic countries. For example, detection of PCR
products, historically performed by radioactively labeled
Southern blot hybridization, can be performed now by
rapid DNA Detection Test Strips (Figs. 1a, 2a) [16]. The
methods used for the detection of PCR products differ
enormously with regards to sensitivity, time consump-
tion, required equipment, costs, and reliability (Table 1).
Although there is now extensive experience on PCR for
lymphatic filariasis on blood and on vector samples,
there still a great need to improve the robustness of the
assays and to standardize the methods. The recent de-
velopments hold the promise that PCR-based detection
of lymphatic filariasis is suitable to be employed in en-
demic countries in the framework of the Global Pro-
gramme for the Elimination of Lymphatic Filariasis.
Hands-on time Costs equipment Remarks Reference
per test (US $)
20 min >1500/<0.05 Reliable, toxic [9, 14, 27]
chemicals
2h >1500/<0.2 Toxic chemicals [6, 8]
1h >1500/<0.1 Convenient for [11, 12]
large numbers
5 min –/1.0– Reliable, almost [16]
no training
required

Acknowledgements We thank Dr. T. Supali for the supply of
B. timori samples and Dr. M. Wilson and H. Baidoo for partici-
pation in the vector studies. P.F. was supported by the scholarship
program ‘‘infectiology’’ of the BMBF and by the UNDP/World
Bank/WHO-TDR. D.B. was supported by the UNDP/World
Bank/WHO-TDR and DFID.
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