DEVELOPMENTS IN FILARIASIS RESEARCH

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� Springer-Verlag 2002

Abstract PCR-based diagnostic assays are promisingtools for the monitoring and evaluation of the GlobalProgramme for Elimination of Lymphatic Filariasis.Sensitive and specific assays have been described for thedetection of Wuchereria bancrofti, Brugia malayi, orBrugia 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 laterstages of control programs when infection rates of hu-mans and vectors are low, and large numbers of samplesmust be examined.

Keywords Filariasis Æ Diagnosis Æ Blood ÆSputum Æ Mosquitoes

Introduction

To establish sensitive and specific PCR-based diagnos-tics for lymphatic filariasis different target DNA repeatshave been identified for the filarial parasitesWuchereriabancrofti and Brugia species. In Brugia a tandemly re-peated sequence of about 320 bp (Figs. 1, 2) designatedHhaI repeat can be found in 30,000 copies (10% of thegenome) [20]. Similar highly repeated sequences appear

to be absent in W. bancrofti, but several moderatelyrepeated sequences have been identified [8, 19, 26, 28, 30,32]. Based on one of these sequences termed the SspIrepeat, the oligonucleotide primers NV-1 and NV-2 havebeen developed and used for most PCR diagnosticstudies of W. bancrofti. Using these primers a PCRproduct 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 inBrugia parasites. In addition to these PCR targets, DNAsequences encoding for rRNA or spacer regions betweenrRNA genes can be used for PCR amplification of fi-larial DNA. These target sequences are especially ofvalue when information about more highly repeatedsequences is not available [10, 21].

Although selection of target sequences is an impor-tant step in the establishment of PCR assays, DNAextraction, optimization of PCR conditions and cy-cling, and convenient and sensitive detection of PCRproducts are critical. Assays must be adapted depend-ing on the sample from which filarial DNA should beamplified.

Detection of parasite DNA in blood samples

The availability of a very sensitive and convenient cardtest that detects W. bancrofti circulating antigen [29]leaves detection of W. bancrofti infection in humans byPCR with only a limited field applicability. However, thetest for circulating adult worm antigen remains positivefor some time although microfilaria densities havedropped after treatment while PCR is only positive ifmicrofilariae or free DNA derived from them is presentin the blood. Our observations show that detectable freeDNA comes from dying microfilariae and is only stablefor a few days in human blood. Some studies requireinformation on the presence of microfilariae, and PCRon blood samples can be a very sensitive alternative tothe conventional detection of microfilariae using par-asitological methods. In contrast to W. bancrofti, no

Med Microbiol Immunol (2003) 192: 3–7DOI 10.1007/s00430-002-0152-z

P. Fischer (&)Bernhard Nocht Institute for Tropical Medicine,Bernhard-Nocht-Strasse 74,20359 Hamburg, GermanyE-mail: Pfischer@bni.uni-hamburg.deTel.: +49-40-42818486Fax: +49-40-42818400

D. BoakyeNoguchi Memorial Institute for Medical Research,University of Ghana,Legon, Accra, Ghana

J. HamburgerKuvin Center for the Study of Infectious andTropical Diseases,Hebrew University of Jerusalem, Israel

sensitive and specific antigen test is available for thedetection of B. malayi and B. timori infection in humans.Therefore PCR on blood samples (and in the future alsoon 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 subsequentdigestion of white blood cells and microfilariae usingproteinase K [30]. This method has been used to prepareDNA from infected individuals with low microfilariadensities but with free parasite DNA and DNA in dayblood of microfilaremic persons since free DNA tends toadhere to cells [12]. For convenient sample collection,preservation, and subsequent DNA extraction smallamounts of venous or capillary blood can be collectedon 3MM Whatman filter paper, dried, and stored atambient temperature. DNA is then extracted by a simpleboiling method using Chelex 100 resin to bind PCRinhibitors [16].

DNA of nocturnally periodic B. malayi can be de-tected by PCR–enzyme-linked immunosorbent assay in200 ll of night or day blood samples with a higher or atleast an equal sensitivity compared to the filtration of1 ml night blood [12]. The PCR assay established for thedetection of B. malayi is also used to detect microfilariaeof B. timori since these two species appear to have anidentical HhaI repeat [31]. It was possible to detect asingle microfilaria of B. malayi or of B. timori with thesame assay (Fig. 1). An ongoing study in Indonesiashows that the HhaI repeat of B. timori is a suitabletarget for its PCR-based detection in human and vectorsamples (P. Fischer, T. Supali, unpublished results).

A PCR pool-screening approach would be needed todetermine the presence of microfilariae within a com-munity. In a pilot study we collected finger-prick bloodon filter paper and pooled four blood spots of 15 ll fromfour noninfected individuals with one 15-ll night bloodspot of an infected person with low microfilaria density

(40–80 microfilariae/ml). Two of three pools were posi-tive by PCR, and it can be assumed that the negativepool contained no microfilaria (Fig. 2). Although inlarger amounts of blood free parasite DNA can be de-tected by PCR [12], free DNA can be rarely detected insmall blood spots (P. Fischer, T. Supali, unpublishedresults). Other studies show that free DNA can be de-tected only using a nested PCR approach [7], but thisbears a high risk of contamination and may limit itsapplication in many laboratories [5].

Detection of parasite DNA in sputum

There is a need to establish PCR assays based on humanmaterial that can be collected noninvasively such asurine or sputum. Lucena et al. [18] reported the detec-tion ofW. bancrofti DNA in urine by PCR. However, itis still not known whether this method is sensitive andreliable enough for field application. Sputum PCR fordiagnosis of lymphatic filariasis is at this time still in itsstage of development and validation but is a promisingnew tool.

Sputum PCR aims at adding further logistic advan-tages to methods that are based on daytime samplecollection, such as antigen tests [29], and PCR assays fortesting daytime blood [12]. All blood-based diagnostictests share the problematic aspects of blood collection.By comparison, the collection of sputum is noninvasive,widely acceptable, can be carried out by village workerswith minimal training, enables storage and shipment ofsamples at ambient temperature, can be performed overa relatively long period of time for maximal represen-tation of the target population, and is also relativelyinexpensive.

The putative presence of DNA of lymphatic filariaein sputum derives support from a number of consider-ations. (a) These largely nocturnally periodic parasites

Fig. 1 Detection of the HhaI repeat in B. malayi and in B. timoriby 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 ofbiotinylated PCR products using a digoxygeninated DNA probeand DNA Detection Test Strips; T test line; C control line; Bagarose gel; M molecular weight marker

Fig. 2 Detection of the HhaI repeat of B. malayi in pools of bloodspots by PCR. Lane 1 One 15 ll blood spot from a person with 1.2microfilariae (mf) per 15 ll (80 mf/ml) and four negative bloodspots; 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 bloodspot from a person with 0.6 mf per 15 ll (40 mf/ml) and fournegative blood spots; lanes 4–5 pools of five negative control bloodspots. A Detection of biotinylated PCR products using adigoxygeninated DNA probe and DNA Detection Test Strips; Ttest line; C control line; B agarose gel; M molecular weight marker

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reside during the day in the microvasculature of thelungs, readily available for clearance. Perhaps nematodelarval migration from the pulmonary blood vessels toalveoli (as with ascarids, hookworms, etc.) is clearancetaken several steps further in evolution along the sameanatomical route. (b) The size of microfilariae (250–300 lm in length) is not conducive to simple clearanceby phagocytosis, thus clearance of microfilariae throughthe lungs is a logical alternative. (c) Tropical pulmonaryeosinophilia 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 sputumPCR was initially demonstrated by testing a few diur-nally collected sputum samples from patients in theNorth Coast Province, Kenya [1]. A more extendedstudy then followed by testing sputum samples fromKenyan patients exhibiting parasitological and/or clini-cal evidence of lymphatic filariasis and from endemicnormals [2]. Collection of sputum in 0.2 M EDTA in-hibits bacterial growth and enables storage at ambienttemperature for a several weeks [17]. A very simple al-kaline DNA extraction that does not involve enzymesand separation matrices was adapted for sputum PCR.PCR primers employed in this study were derived from along 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), thefirst one in parasites (I. Abbasi, R. Ramzy, S.A.Williams, J. Hamburger, submitted). S/MARs are highlyrepresented (20,000 copies or more per haploid genome)in the genome of eukaryotes (reviewed in [4]), and suchhigh representation offers high detection sensitivity toPCR assays employing corresponding primers. This is anew approach for seeking suitable primers for identify-ing eukaryotic parasites. The primers so far employedwith 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 34sputum samples collected from patients with proveninfection, 32 (94%) were PCR positive, but those withsymptoms were 100% PCR positive suggesting that insymptomatic patients microfilaria clearance is morepronounced. Testing pools of sputum samples by PCR

(1 sample from an infected individual plus 14 samplesfrom uninfected ones) has also been carried out [2].

Standardization of sputum collection and large-scalevalidation of sputum PCR are now in progress inKenya. Development of sputum PCR for diagnosis ofbrugian filariasis is of particular importance but stillrequires identification of suitable PCR primers. TheHhaI 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 thatLDR1 homologue is present in Brugia DNA. Its iden-tification may enable the design of Brugia-specificprimers 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 theirrespective vectors has been an essential component indetermining areas at risk of infection, the transmissionpotential of vectors, and also a direct indication thattransmission is occurring. Traditionally this has beendone by dissecting the mosquito vectors and examiningthem under the microscope to morphologically identifythe parasites. This method is time consuming, labor in-tensive, and prone to observer bias, particularly so wheninfection levels in the vector populations are very low.The development of an efficient, rapid, sensitive, specific,and cost-effective tool to replace the classical dissectionmethod is therefore necessary to monitor and evaluateintervention programs. The development of a PCR-based pool-screening method paves the way for thedevelopment of such a diagnostic tool.

A PCR assay amplifying 380- and 650-bp fragmentsofW. bancrofti DNA was initially developed for identi-fication of infected mosquitoes [8]. This method yieldedlow sensitivity due to PCR inhibitors from mosquitomaterial and lacked in test convenience since PCRproducts 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] demonstratedthe possibility of using the PCR method to detect W.bancroftiDNA in pools of mosquitoes and employed theSspI repeat derived primers (NV-1 and NV-2) for im-provement in sensitivity. Detection was carried out inpools of 50 Aedes polynesiensis heads, and three DNAextraction protocols were compared for this purpose [6].The best results were obtained when a boiling andfreezing step was included. All recent studies except forone [28] have also used these primers to detect W. ban-crofti in mosquitoes.

Further improvement in test sensitivity by Nicolaset al. [23] enabled the detection of a single mosquitoinfected with one or two microfilariae of W. bancroftiamong 20–50 mosquitoes or one L3 in 50–100

Fig. 3 PCR pool screening to detect one W. bancrofti L3 indifferent pools of adult A. gambiae s.s. heads using the dynabeadpurification system and the SspI repeat as target. Agarose gel; Mmolecular weight marker; lanes 1, 2 100 mosquitoes; lanes 3–4 75mosquitoes; lanes 5, 6 50 mosquitoes; lanes 7, 8 25 mosquitoes; lane9 10 mosquitoes; lane 10 positive control; lane 11 negative control

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A. polynesiensis. The PCR product was detected by acharacteristic band on an ethidium bromide stainedagarose gel and other more sensitive detection methodsfor PCR products may even increase this sensitvity.However, the assay was as sensitive as the dissection ofmosquitoes infected withW. bancrofti. The PCR methodwas further evaluated by Ramzy and coworkers [14, 27]on field-collected Culex pipiens from Egypt (see [14]). Ithas also been evaluated on C. quinquefasciatus [13] andon Anopheles punctulatus [3]. Recently Farid et al. [9]have reported the potential for using the pool screeningin estimatingW. bancrofti infection in pools of C. pipiensfrom two villages with different prevalence rates of hu-man filariasis. A drawback to the technique has been theinconsistency 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 thisdrawback [3, 11, 22].

Currently the PCR pool-screening method has beendeveloped to detect one infective W. bancrofti larva in apool 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., animportant vector of W. bancrofti in Africa. For the PCRdetection to be cost effective there is the need to increasethe pool size and to improve theDNA extractionmethod.One way of improving the extraction to obtain theparasite DNA of interest is to use magnetic bead capturesystem. We have used this procedure described below toincrease the pool size to 75–100 mosquitoes (Fig. 3).

The DNA was extracted according to the protocol ofZimmerman et al. [33] and then purified using an equalvolume of 2.5 lmol labeled NV-1 capture primer andDynabead binding buffer (Dynal MPC -S, Oslo, Nor-way) according to the instructions of the manufacturer.Following denaturation, capture primer annealing, andwashing of the Dynabeads the DNA solution was in-cubated overnight with the magnetic Dynabead parti-cles. After several washing steps on the Dynal magneticparticle concentrator the target DNA was separatedfrom the beads and removed into a new tube. Of thesupernatant 2 ll was used in each PCR.

The Dynabead purification method has recently beenused to detect W. bancrofti in members of the A. gam-

biae s.s. from areas in Ghana where mass treatment withivermectin/albendazole is planned. Infection rates will beestimated using the algorithm of Katholi et al. [15], andthe results will be compared with those obtained fromthe classical dissection. The PCR has been shown to beeffective for detectingW. bancrofti in various mosquitovector species and could be used to monitor the outcomeof intervention measures. In addition, no differences inDNA preparation of mosquitoes infected with W. ban-crofti or with Brugia species have been reported. Usingthe HhaI repeat as target for PCR B. malayi andB. 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 forthe identification of endemic areas and for the moni-toring of intervention programs in humans and vectors[25]. For the latter purposes examination of pooledsamples is most efficient. In areas with high microfilariadensities in humans and high infection rates of vectorsparasitological methods are superior to PCR-basedtechniques, but the opposite is true for areas with lowendemicity or advanced control programs. PCR meth-ods have been improved over the last decade and arenow more suitable to be employed in laboratories ofendemic countries. For example, detection of PCRproducts, historically performed by radioactively labeledSouthern blot hybridization, can be performed now byrapid DNA Detection Test Strips (Figs. 1a, 2a) [16]. Themethods used for the detection of PCR products differenormously with regards to sensitivity, time consump-tion, required equipment, costs, and reliability (Table 1).Although there is now extensive experience on PCR forlymphatic filariasis on blood and on vector samples,there still a great need to improve the robustness of theassays and to standardize the methods. The recent de-velopments hold the promise that PCR-based detectionof 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.

Table 1 Comparison of methods for the detection of PCR products in the diagnoses of lymphatic filariasis

Method Specificity Sensitivity Duration Hands-on time Costs equipmentper test (US $)

Remarks Reference

Agarose gelelectrophoresis

Size specific >10 ng 2 h 20 min >1500/<0.05 Reliable, toxicchemicals

[9, 14, 27]

Southern blot Sequencespecific

Approx. 1 ng 1 day 2 h >1500/<0.2 Toxic chemicals [6, 8]

Enzyme-linkedimmunosorbentassay

Sequencespecific

100 pg–5 ng 5 h 1 h >1500/<0.1 Convenient forlarge numbers

[11, 12]

DNA DetectionTest Strips

Sequencespecific

Approx. 5 ng 30 min 5 min –/1.0– Reliable, almostno trainingrequired

[16]

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Acknowledgements We thank Dr. T. Supali for the supply ofB. timori samples and Dr. M. Wilson and H. Baidoo for partici-pation in the vector studies. P.F. was supported by the scholarshipprogram ‘‘infectiology’’ of the BMBF and by the UNDP/WorldBank/WHO-TDR. D.B. was supported by the UNDP/WorldBank/WHO-TDR and DFID.

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