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 employed for pool screening, which is important in the later stages of control programs when infection rates of humans 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 diagnostics for lymphatic filariasis different target DNA repeats have been identified for the filarial parasitesWuchereria bancrofti and Brugia species. In Brugia a tandemly repeated 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: Pfi
[email protected] 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 filarial 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 important step in the establishment of PCR assays, DNA extraction, optimization of PCR conditions and cycling, and convenient and sensitive detection of PCR products are critical. Assays must be adapted depending 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 parasitological methods. In contrast to W. bancrofti, no
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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 detection of brugian filariasis. Efficient DNA extraction is crucial for any PCR assay. A simple and inexpensive method for DNA preparation 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 detected 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 community. 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 positive 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 detected 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 detected 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 detection 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 advantages 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 representation 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 considerations. (a) These largely nocturnally periodic parasites
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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 microfilariae undergoing immune-clearance in the lungs (reviewed in [24]). Expectoration of microfilarial constituents is not unlikely under microfilarial clearance. The ability to detectW. bancrofti DNA by sputum PCR was initially demonstrated by testing a few diurnally 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 clinical evidence of lymphatic filariasis and from endemic normals [2]. Collection of sputum in 0.2 M EDTA inhibits bacterial growth and enables storage at ambient temperature for a several weeks [17]. A very simple alkaline 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 genome. LDR1 was later shown to be a region of attachment 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 identifying eukaryotic parasites. The primers so far employed with similar results, are AccI primers amplifying a 254bp-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
(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 LDR1based 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 identification may enable the design of Brugia-specific primers for diagnosis of brugian filariasis by sputumPCR. 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 intensive, 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 PCRbased 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 identification 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 individual mosquitoes and would be for monitoring purposes 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 improvement 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. bancrofti 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
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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 human filariasis. A drawback to the technique has been the inconsistency sometimes observed, leading to false negatives presumably due to the presence of PCR inhibitors. Coamplification of parasite DNA together with an internal 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. polynesiensis, and A. punctulatus. No study has yet been reported 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, Norway) according to the instructions of the manufacturer. Following denaturation, capture primer annealing, and washing of the Dynabeads the DNA solution was incubated overnight with the magnetic Dynabead particles. 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-
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. bancrofti 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 differentiated 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 diagnosis of lymphatic filariasis, but more importantly for the identification of endemic areas and for the monitoring 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 methods 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 consumption, 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 developments hold the promise that PCR-based detection of lymphatic filariasis is suitable to be employed in endemic countries in the framework of the Global Programme 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 equipment per test (US $)
Remarks
Reference
Agarose gel electrophoresis Southern blot
Size specific
>10 ng
2h
20 min
>1500/1500/1500/
