A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.


Introduction
Onchocerciasis, or River Blindness, is a neglected tropical disease caused by the parasitic worm Onchocerca volvulus. The parasite is transmitted to humans through exposure to repeated bites of infected black flies of the genus Simulium. The disease is a major public health concern, and has severe social and economic impact. Recent estimates indicate that, more than 30.4 million people are infected, mostly in sub-Saharan Africa [1,2]. Over 700,000 people are visually impaired and another 265,000 are blinded by the disease [3]. There is no vaccine against infection or suitable macrofilaricidal drug that kills the adult stage of O. volvulus. Current control is based on annual or semi-annual distribution of the larvicidal compound ivermectin (Mectizan, Merck) to the population irrespective of infection status [4][5][6]. In the absence of an adulticide, it is recommended that these mass drug administration (MDA) campaigns should be continued for 10-15 years [7].
MDA programs have now progressed for several years in many areas, and careful monitoring of infection levels in human populations, as well as vectors, is required to evaluate their success, certify elimination and guide the decision to stop MDA.
Definitive diagnosis of infection with O. volvulus in humans involves identification of subcutaneous nodules or observation of microfilariae in skin snips using microscopy. The detection of microfilariae in skin can be a challenge when parasite densities are low, which is often the case when MDA programs are underway. Several serological methods exist involving antibody detection to O. volvulus-specific antigens. The most widely used assays are based on the detection of IgG4 responses to the Ov-16 antigen in children [8][9][10][11]. Of all the methods developed thus far for diagnosis of infection in humans, the highest levels of sensitivity have been achieved in skin snip/scratch analyses using the polymerase chain reaction (PCR) targeting the O-150 repeat sequence [12][13][14]. Infection rates in black flies are rapid and sensitive indicators of the change in community microfilarial load that results from ivermectin distribution, and correlate well with the percentage coverage of the community [7]. Importantly, they are also an important indicator of when MDA is succeeding in breaking transmission of O. volvulus. In addition, from logistical and ethical perspectives, monitoring infections in the vector offers some advantages over repeated blood examinations of the human population [15,16]. For detection of O. volvulus infection in black flies, the World Health Organization (WHO) recommends the use of PCR-based methods [7]. To date, these assays have been performed using the O-150 repeat sequence identified more than 20 years ago [17][18][19][20][21][22], where the amplification products are subsequently detected by several methods including an enzymelinked immunosorbent assay (PCR-ELISA) [23][24][25][26].
In sub-Saharan Africa, cattle are frequently infected with Onchocerca ochengi, a species that exclusively parasitizes Bovidae. This is the closest extant relative of O. volvulus and is transmitted in West Africa by the same species complex of black fly vectors, Simulium damnosum sensu lato [27]. Discrimination between these two species requires an additional step of hybridization of the PCR amplified products with an O. volvulus-specific DNA probe [17,19,28,29]. Since the complexity of a test can be a technical barrier, a simpler method for the specific detection of human parasites in the vector would be a significant advance. In addition for low-resource settings, PCR can be a challenge as it requires skilled personnel and expensive equipment [30]. Therefore a new molecular method for the detection of O. volvulus that circumvents some of the current limitations would be a useful tool to aid onchocerciasis control and elimination efforts [31].
Loop-mediated isothermal amplification (LAMP) is an alternative technique which amplifies DNA with high specificity, sensitivity and rapidity under isothermal conditions [32]. The LAMP reaction includes two sets of primers that hybridize to six sites on the target DNA, and a third set of primers (loop primers) to accelerate the reaction [33]. The mixture of stem-loops containing alternately inverted repeats of the target sequence and cauliflower-like structures that are generated result in exponential amplification of the target sequence (.10 mg,.506 PCR yield) [32][33][34]. Using three primer sets recognizing eight sites in the target DNA engenders the specificity to discriminate between genomic DNA at both genus and species specific levels [35,36]. In recent years this technology has been explored for the diagnosis of several infectious diseases including those caused by parasitic protozoa [37,38] and the filarial parasites Brugia malayi [39], Wuchereria bancrofti [40] and Loa loa [41,42]. The simplicity, rapidity, and versatility in readout options available for LAMP, offer a distinct advantage over other molecular diagnostic methods. LAMP test kits for use in resource-limited settings are now commercially available for the detection of Mycobacterium tuberculosis complex [43,44] and human African trypanosomiasis [45].
In the present study we report on the identification of a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), and the development of a simple, single-step, LAMP assay that easily distinguishes between O. volvulus and O. ochengi DNA. Our results demonstrate that the test represents a significant technical advance, and has the potential to be used as a new field tool for surveillance of parasite transmission and evaluation of MDA programs for onchocerciasis.

Reagents
O. ochengi DNA was extracted from adult worms obtained from cattle skin nodules after normal processing at the Ngaoundéré abattoir, Adamawa Region, Cameroon. L. loa DNA was prepared from infective stage larvae isolated from Chrysops silacea collected in the Southwest Region of Cameroon. Genomic DNA was extracted using DNAzol reagent (Invitrogen) according to the manufacturer's instructions. Onchocerca volvulus genomic DNA was prepared from adult female worms as described [46]. Bovine DNA and human DNAs were obtained from Millipore, USA.

Black flies
Uninfected, laboratory reared female Simulium vittatum were obtained from the Black fly Rearing and Bioassay Laboratory, University of Georgia, USA. Pools containing varying numbers of black flies (50, 100, 150 and 200 each) were prepared according to established protocols [21,23].

Spiking and DNA extraction
Each pool of black flies was placed in a 1.5 mL micro centrifuge tube and the insects were crushed in 500 mL extraction buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 100 mg/mL of proteinase K) using a blunted glass pipette. An additional 500 mL extraction buffer containing either no DNA, or purified O. volvulus genomic DNA
For comparative analysis of sigma GST genomic sequences, OvGST1a and OvGST1b and the O. ochengi homologue g09064 were aligned over the complete gene sequence (total distance, 3,870 bp) using Kalign [54,55] [50]. Regions specific for O. volvulus were identified in OvGST1a and LAMP primers were designed to target the gene using Primer Explorer V4 [http://primerexplorer.jp/e/]. Two sets of primers comprising two outer (F3 and B3), and two inner (FIP and BIP) were selected. FIP contained F1c (complementary to F1), and the F2 sequence. BIP contained the B1c sequence (complementary to B1) and the B2 sequence. Additional loop primers, forward loop primer (FLP) and backward loop primer (BLP) were included in the reaction.
The outer LAMP primer pair F3 and B3 was also used for specific amplification of OvGST1a by PCR. PCR primers for amplification of actin were as previously described [39]. The mixture was incubated at 63uC for 60 min, then heated at 80uC for 2 min to terminate the reaction. Reactions were carried out using either a Loop Amp Realtime Turbidimeter (LA-320c, Eiken Chemical Co, Japan) or a 2720 Thermocycler (Applied Biosystems, USA) set at a constant temperature for colorimetric detection. A positive reaction was defined as a threshold value greater than 0.1. Turbidity data were analyzed using the LA-320c software package that reports when the change in turbidity over time (dT/dt) reaches a value of 0.1, which we then assigned to be the threshold time (Tt). For determination of amplification measured by color change (purple to sky blue), 0.15 mL of 120 mM hydroxy naphthol blue (HNB, Sigma-Aldrich Inc, St. Louis, MO, USA) was added to the reaction mixture. All experiments were performed in duplicate at least 3 times.

PCR assay
LAMP primers B3 and F3 were used to PCR amplify OvGST1a in 25 mL reactions containing 3 mL DNA template, 0.2 mM of each primer, and 1.25 U of Taq DNA polymerase in 16 standard buffer (New England Biolabs) containing 3.5 mM MgCl 2 , 0.2 mM and 0.2 mM dNTP each. All reactions were denatured once at 94uC for 5 min followed by 35 cycles of the following cycling conditions: 30 s at 94uC, 1 min at 53uC, 1 min at 72uC, and a final extension for 5 min at 72uC using a Gene Amp PCR system 9700 (Applied Biosystems). PCR products were visualized by UV transillumination in a 1.5% agarose gel after electrophoresis and staining with ethidium bromide. As a positive control for the presence of intact DNA, a 244 bp actin fragment was PCR amplified as described [40]. The key for protein sequence accession numbers and organisms displayed in the tree is as follows

Results
During manual curation of gene predictions in the O. ochengi genome (http://www.nematodes.org/genomes/onchocerca_ochengi), it was noted that this species has one copy of the glutathione S-transferase-1 gene (OoGST1), whereas O. volvulus has two copies [56]. Phylogenetic analysis using protein sequences demonstrated that although two additional gene models containing GST sigma-like domains are present in the O. ochengi genome, these are unrelated to the two OvGST1 paralogues and cluster at different branches of the tree (Fig. 1). Indeed, the ''GST1'' group [comprising OvGST1a, OvGST1b and OoGST1 (CDS t09064)] form a highly distinctive clade, which is distant not only from insect and mammalian sigma GSTs, but also from those of other nematodes, including filarial representatives and Ascaris suum (an additional clade III nematode) (Fig. 1). Intron/exon sequence and gene structure were found to be highly conserved within the ''GST1'' group (Fig. 2). Overall nucleotide identity was.90% for all exons and introns between OoGST1 and both of the O. volvulus GST1 genes. However, OvGST1b is most similar to OoGST1 at 98% overall identity, in comparison to OvGST1a at 96% identity ( Fig. 1 and Fig. 2). The three major differences between the genes comprised insertions in intron 3 of OvGST1a.
Based on the phylogenetic tree and comparative sequence analyses, several primer sets targeting OvGST1a and/or OvGST1b were evaluated (data not shown). Assays were performed in the temperature range 60-65uC for up to 90 minutes using various concentrations of MgSO 4 (4, 6, 8, and 10 mM) and primers (0.1, 0.2, and 0.4 mM F3 and B3; 1, 1.5, 2, and 4 mM FIP and BIP; and 0.5, 1, and 2 mM FLP and BLP), as well as varying the primer sequences. The optimum incubation condition was established as 63uC for 60 min in a buffer containing 4 mM MgSO 4 , followed by heating at 80uC for 2 min to terminate the reaction. In accordance with the sequence analysis, OvGST1a was revealed as the best target (data not shown). Primer sets (Fig. 3A and Fig. 3B) targeting OvGST1a were designed after optimization and used for specificity and sensitivity studies.
Specificity of this primer set was determined in LAMP, using a real time turbidimeter (Fig. 4A) and colorimetric detection (Fig. 4B), to monitor amplification of genomic DNA from O. volvulus, O. ochengi, or a related human filarial parasite, Loa loa. Bovine, human, and black fly genomic DNAs, and non-template controls were also included for comparison. Turbidity reached a threshold value of 0.1 in approximately 45 minutes when 1 ng O. volvulus DNA was added to the reaction, whereas no turbidity was observed within the time interval examined (90 minutes) when the same amount of heterologous DNAs from O. ochengi, L. loa, mammal or black fly was used (Fig. 4A). Similar results were observed using the more simplified colorimetric detection method, where a color change (purple to blue) was only evident when O. volvulus genomic DNA was present (Fig. 4B). Conversely, in the absence of template or primers, no reactions were observed when using either turbidity or color change as the readout (Fig. 4A and  4B).
Specificity studies were also performed by PCR amplification of OvGST1a using primers F3 and B3 (Fig. 4C). A 200 bp fragment of the expected size was obtained when O. volvulus genomic DNA was used as a template, whereas no product was observed from samples containing heterologous DNA or no template. The integrity of the various DNAs was confirmed in PCR experiments using primers designed to amplify a conserved actin gene. A single amplification product of the correct size (244 bp) was observed in all cases (Fig. 4D).
To determine and compare the detection limits of LAMP and PCR, ten-fold serial dilutions of O. volvulus genomic DNA ranging from 0.001-1.0 ng were amplified (Fig. 5A-C). Both amplification methods were able to detect levels as low as 0.01 ng, which is equivalent to 1 / 10 th of a single microfilaria. In the case of LAMP a positive result was evident within one hour (Fig. 5A).
Since the goal is to use the LAMP assay to evaluate infection in the vector, pools of uninfected, laboratory-reared black flies were spiked with 0.001-1.0 ng O. volvulus genomic DNA, and total genomic DNA was then isolated using a commercially available DNA extraction kit or by boiling. Samples from each pool and extraction method were then used as templates for amplification of OvGST1a in LAMP and PCR reactions ( Table 1). Consistent with previous results using highly purified DNA as template, LAMP was positive in samples prepared from an insect pool containing 50-200 black flies spiked with 0.1 ng O. volvulus DNA (equivalent to a single microfilaria) when DNA was purified using a commercially available kit, or extracted in a more crude fashion by boiling. PCR was less effective following crude extraction with a pool size limit of 150 black flies. At the 0.01 ng level using kit purified material, LAMP efficiently amplified OvGST1a in pool sizes up to 150 black flies, whereas for PCR the pool size limit was 50 insects. When boiling was used to extract DNA, a positive signal was obtained for LAMP at a ratio of 0.01 ng target DNA in 100 insects, while the limit for PCR was 0.01 ng DNA in 50 insects. These results demonstrate the ability of the LAMP assay to withstand the inhibitory effects of components present in the purified or crude black fly extracts without severely affecting sensitivity.

Discussion
In recent years there has been significant progress in the control of onchocerciasis by treating whole populations with repeated, semi-annual (Latin America) or yearly (most African foci) cycles of ivermectin [57]. Several agencies are involved in these activities for example, the African Programme for Onchocerciasis Control (APOC), and the Onchocerciasis Elimination Program for the Americas (OEPA). Surveys of Simulium vectors are recommended by WHO to determine if transmission has been interrupted and to certify that elimination of the parasite has been achieved [7]. Previous studies have shown the value of molecular xenodiagnosis(detection of parasite DNA in insects by DNA amplification methodologies) as a tool for assessing changes in parasite prevalence rates in endemic populations after MDA [12,17,21,23,58]. This method requires collection of representative samples of insects, isolation of total DNA from insect pools, amplification of parasite-specific DNA sequences, and detection of the amplified product. Currently, PCR pool screening of large numbers of flies is employed since infection levels are likely to be low or non-existent in treated areas. There is a limit to the number of flies in each pool, since the DNA polymerases used in PCR reactions are highly sensitive to inhibitors present in insect extracts. Currently, either silica-purified DNA or oligonucleotide capture of O. volvulus genomic DNA from homogenates of insects is used to reduce the amount of inhibitors carried over into the reaction [23]. Another approach involves reducing the insect biomass by limiting the analysis to insect heads alone. This will also reveal the prevalence of flies carrying infective-stage larvae (L3) and therefore provide an accurate assessment of transmission, and high-throughput methods for collecting black fly heads have been developed for this purpose [22,25]. Current OEPA guidelines require that the prevalence of flies carrying L3s be less than 1/2000 in every sentinel community for transmission to be interrupted [59], which necessitates surveying approximately 6000 flies from each area to state with 95% confidence that the prevalence of infective flies is in this range [23].
In sub-Saharan Africa where cattle-biting S. damnosum s.l. flies and zebu cattle are present, O. ochengi infections are common in the vector population [60,61]. Based on the presence of microfilariae, the prevalence in cattle is as high as 66-71% in some areas [62]. The parasite is extremely closely related to O. volvulus, as determined by phylogenetic distance [63] and natural history [64]. Indeed, it has been hypothesized that O. volvulus diverged from O. ochengi as recently as 5,000 years ago during the domestication of cattle in sub-Saharan Africa [65]. The routinely used O-150 diagnostic marker for O. volvulus clusters with other Onchocerca species, thereby hampering species discrimination [66].
In the present study we identified a gene (OvGST1a), encoding a glutathione S-transferase, as an alternative biomarker for O. volvulus infection. GSTs (EC 2.5.1.18) are an ancient and diverse superfamily of multifunctional proteins. Three different classes of GST (OvGST1-3) have been isolated and characterized from O. volvulus [67,68]. The OvGST1a and OvGST1b isoforms (differing in only 10 amino acids) [56,69] are unique sigma-class GSTs that encode an extracellular enzyme located in the outer zone of the hypodermis at the host-parasite interface, where they are thought to influence host inflammatory and immune cells due to their GSH-dependent prostaglandin D Synthase activity [56,67,70]. GSTs are present in all the developmental stages of the parasite and have been pursued as potential vaccine/drug targets [70]. The presence of two GST1 paralogues in O. volvulus suggests that the GST1 gene underwent a duplication event following the speciation of the human parasite from its bovinespecific sister. We evaluated the suitability of OvGST1b (data not shown) and OvGST1a for diagnosis of O. volvulus infection using both LAMP and PCR methods. High levels of specificity were achieved in OvGST1a-based LAMP and PCR assays. LAMP primers amplified O. volvulus DNA but not DNA isolated from the closely related filarial parasites O. ochengi or L. loa, or from human, bovine or black fly. LAMP primers F3 and B3 showed a similar specificity profile when used in PCR reactions, highlighting the versatility of this target for molecular diagnostic studies.
High levels of specificity and sensitivity can be achieved in LAMP because the amplification reaction involves four specific oligonucleotide primers that anneal to six distinct regions within the target sequence [32]. The addition of loop primers may further improve performance [34]. We observed comparable levels of sensitivity (0.01 ng), equivalent to 1 / 10 th of a single microfilaria [71], using either LAMP or PCR to amplify OvGST1a when highly purified DNA was used as template. We would therefore predict that the assays would permit detection of a single infective stage larva given that they are considerably larger in size. However, LAMP was more efficient than PCR in detecting O. volvulus DNA recovered from black fly material (0.01 ng in 150 insects within 60 minutes). This is likely due to the fact that black flies contain a number of biological substances that inhibit the polymerases used in PCR which cannot be removed completely during classical extraction protocols. The most efficient method used to circumvent this problem involves paramagnetic bead Table 1. A comparison of LAMP and PCR methods to detect varying amounts of genomic DNA isolated from pools of black flies using different methods. purification, but it is expensive [25]. Other studies have also shown superior tolerance of LAMP tests for biological substances [72][73][74]. Furthermore unlike PCR, LAMP proved effective even when DNA was extracted using a simple boiling method, rather than using commercially available kits that add a significant cost to the process (as well as time and effort). This is a significant finding representing an important technical advance, and emphasizes the usefulness of the LAMP technique as a surveillance tool for mass screening of infected vectors. In addition, recent estimates suggest that diagnostic LAMP tests are significantly cheaper than PCR. The estimated cost of a W. bancrofti LAMP test is $0.82 compared with more than $2.20 for PCR [40]. Other distinct advantages of LAMP over PCR include its operational simplicity and isothermal nature. In PCR, thermal cycling is required to denature the template, anneal primers and extend the amplicon. LAMP employs Bst DNA polymerase, which provides both strand displacement and target amplification at a single temperature in a simple heat block or water bath at 60-65uC [32]. Rapidity and versatility in readout options also make LAMP a particularly appealing technology. In the present study, real-time turbidity was used for assay design and optimization yielding positive results within 60 minutes, and results were confirmed using the more field-friendly hydroxy naphthol blue [75,76].
All the data on detecting O. volvulus-specific OvGSTa DNA were derived from pools of laboratory reared S. vittatum spiked with purified O. volvulus gDNA. Further work is required to demonstrate that the extraction techniques employed are able to release sufficient template for detection from at least one infected fly in a pool of insects. The current recommendation for the number of flies in a pool, limited by the DNA purification process, is 50 flies for Latin American vectors and 100 flies for African vectors [23]. We anticipate that the OvGSTa LAMP assay will accommodate these pool sizes since the data from DNA-seeded pools (up to 200 insects) indicates that the method is robust and the extraction protocol employed will likely suffice to release measurable DNA target from a single infected black fly.
In summary, we describe a simple OvGST1a-based LAMP diagnostic assay for O. volvulus infection that generates a robust read-out within 60 minutes. The assay has considerable potential as a new field tool for implementation and management of MDA programs for onchocerciasis.