Onchocerca volvulus: comparison of field collection methods for the preservation of parasite and vector samples for PCR analysis.

Onchocerca volvulus: comparison of field collection methods for the preservation of parasite and vector samples for PCR analysis L. Toe,1 C. Back,2 A.G. Adjami,3 J.M. Tang,4 & T.R. Unnasch5 In recent years, methods for the identification of the filarial worm Onchocerca volvulus and its vector, blackflies of the Simulium damnosum complex (S. damnosum sensu lato (s.l.)), based on the amplification of parasite and vector DNA sequences with the polymerase chain reaction (PCR), have been developed. Routine application of these methods requires techniques for sample collection and preservation that are compatible with the limitations of field collection, yet preserve DNA in a form suitable for PCR. Two different methods for sample preservation were evaluated by the field collection teams and the DNA probe laboratory of the Onchocerciasis Control Programme in West Africa. The most successful involved the preservation of material from 0. volvulus and its associated vectors in a dried state on microscope slides. Of over 1200 parasite samples preserved in this manner, more than 93% retained DNA yielding positive results in PCR analysis (1208/1291). Vector material (malpighian tubules and ovaries) preserved in the same manner on the same microscope slides also yielded DNA that was suitable for PCR. Introduction In recent years, DNA-probe techniques using the polymerase chain reaction (PCR) have been devel- oped for the classification of Onchocerca volvulus, the filaria which is the causative agent of oncho- cerciasis (1, 2). The Onchocerciasis Control Pro- gramme in West Africa (OCP) has established a facility in Bouake, Cote d'Ivoire, to use these tech- niques to assist the programme in its epidemiological and entomological monitoring efforts (3). Three types of field samples are collected by the OCP as part of its routine operations. First, infective larvae are taken from adult female Simulium damnosum sensu lato (s.l.) blackflies to estimate their annual transmission potential for 0. volvulus. Second, microfilariae are collected from the skin of infected humans as a part of the epidemiological evaluation of human popu- I Entomologist, Onchocerciasis Control Programme, Bouake, C6te d'lvoire. 2 Coordinator, Applied Research, Onchocerciasis Control Pro- gramme, Bouake, C6te d'lvoire. 3 Research Assistant, Onchocerciasis Control Programme, Bouake, C6te d'lvoire. 4 Postdoctoral Fellow, School of Medicine, Division of Geographic Medicine, University of Alabama at Birmingham, Birmingham, AL, USA. 5 Associate Professor, School of Medicine, Division of Geographic Medicine, University of Alabama at Birmingham, 845 19th Street South, Room 206, Birmingham, AL 35294-2170, USA. Corre- spondence should be sent to Dr Unnasch. Reprint No. 5799 lations. Finally, blackfly specimens (S. damnosum s.l.) are collected to evaluate the vectorial role of sibling blackfly species for 0. volvulus. For all three sample types, field collections are often performed under diffi- cult conditions in sites far from a laboratory facility. In order to use PCR-based methods to classify such samples, it was necessary to have sample collec- tion procedures that met several criteria. First, sam- ple collection has to be rapid and simple enough to be easily carried out by field workers with minimal training. Second, the collection method must pro- duce samples that are stable for prolonged periods of time under ambient conditions, and which can be easily and inexpensively transported to a laboratory facility. Finally, the procedures must be capable of preserving the samples in a form easily recoverable in the laboratory, and they must maintain the DNA contained in these samples in a condition suitable for PCR analysis. The traditional method of preservation of Onchocerca parasite samples for use in molecular biology studies has relied on liquid nitrogen for stor- age (4,5). This is neither simple nor inexpensive, and samples preserved in liquid nitrogen cannot be easily transported. Recently, it has been reported that S. damnosum s.l. adults either preserved in isopropanol or air dried preserve blackfly DNA in a reasonably intact state (6). Similarly, 0. volvulus parasites pre- served in isopropanol may be used to isolate DNA for PCR-based assays (2). These methods offer an attractive alternative to preservation in liquid nitrogen. Bulletin of the World Health Organization, 1997, 75 (5): 443-447 C World Health Organization 1997 443 L. Toe et al. During the past 2 years, these two methods of preservation have been tested by the OCP to inves- tigate their compatibility with PCR, and the results are described below. Materials and methods Field collection and preservation of microfilariae and infective larvae Preservation of larval 0. volvulus specimens in isopropanol was performed following routine dissec- tion of wild caught flies by OCP entomological field teams. Individual adult blackflies were dissected in a drop of normal saline. When larval parasites were detected, they were transferred with a dissecting needle to 1.5-ml microcentrifuge tubes containing approximately 1 ml of isopropanol. While following standard procedures, microfilariae specimens were isolated from skin snips in a similar manner (7), and placed in 1.5-ml microcentrifuge tubes containing approximately 1 ml of isopropanol. Isopropanol- preserved samples were stored at ambient tempera- ture (28-38°C) during transport to the laboratory. When the isopropanol-preserved samples were received, they were centrifuged (14300g) at room temperature (20°C) for lmin, after which most of the supernatant was discarded. The pellet containing microfilariae or larvae was then resuspended in the remaining supernatant, transferred to a microscope slide with a concave well and examined microscopi- cally. Individual parasites were collected with a micropipette and placed in tubes containing 25 tl of TE8.0 (10mmol/l Tris-HCl (pH 8.0) and 1 mmol/l ethylenediaminetetraacetic acid). Preservation of dried parasite and vector speci- mens was also performed after the routine dissection of wild caught flies. The basic steps used for preserv- ing dried larvae and associated vector material are depicted in Fig. 1. When parasite larvae were de- tected, they were separated from insect matter. The blackflies' ovaries and malpighian tubules were also moved to a separate area of the slide, and the re- maining insect debris was discarded. The pools con- taining parasite larvae and the insect ovaries and malpighian tubules were allowed to dry and covered with separate coverslips. Coverslips were sealed at each corner with a drop of nail varnish and stored in collection boxes at ambient temperature (28-38°C) during transport to the laboratory. When received, the location of the parasite was identified, the coverslip removed, and the parasites rehydrated in 25151 of TE8.0. The parasites were then transferred with a dissection needle to a 1.5-ml microcentrifuge tube containing another 25 tli of the TE8.0 solution. Fig. 1. Field collection and processing of dried samples for polymerase chain reaction tests 1. Dissect fly. 2. Isolate parasite larvae, ovaries, and malpighian tubules. Discard insect debris. 3. Air dry. 4. Seal larval and vector material with coverslips. 1. \~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2. © 0 3. 4. WHO973S The dried blackfly material was handled in the same manner. DNA extraction and polymerase chain reaction analysis DNA was extracted from recovered parasite mate- rial following published procedures and used as a template in the Onchocerca-specific 0-150 PCR assay (1). PCR products were separated by elec- trophoresis in a 2% agarose gel (Seakem LE, FMC, Rockland, ME, USA) and visualized by staining WHO Bulletin OMS. Vol 75 1997 8 L )~~~~ (I 444 Onchocerca volvulus: parasite and vector collection methods the gel in a 2 [tg/ml ethidium bromide solution for 15 min. DNA was extracted from the rehydrated black- fly material following published procedures, and PCR amplification of the 238 bp fragment of the NADH dehydrogenase subunit 4 gene and the 910bp internal transcribed spacer of the rRNA gene cluster was carried out as previously described (8, 9). The PCR products were visualized by staining in a 2 [tg/ml ethidium bromide solution following electrophoresis in a 1.5% agarose gel. Results and Discussion A total of 1737 samples were examined. Of these, 425 consisted of microfilariae isolated from skin snips, while 1312 were larvae isolated from wild caught flies. The results obtained with the two methods of sample preservation are summarized in Table 1. Of the microfilarial samples preserved in isopropanol, 48% (73/153) produced detectable PCR products in the Onchocerca-specific 0-150 PCR test. In contrast, 77% (210/272) of the dried microfilarial samples retained DNA yielding positive results on the 0-150 PCR test. Similarly, 74% (217/ 293) of the isopropanol-preserved larval samples produced detectable PCR products, while 98% (998/ 1019) of the dried larval samples did so. This suggests that drying parasites on micro- scope slides is more reliable than preservation in isopropanol. There are two possible reasons for this. First, some of the microcentrifuge tubes containing isopropanol-preserved parasites were accidentally opened during transport, and their contents lost. Second, it was noted that parasites often adhered to the dissection needle or pipette used to transfer them to the isopropanol-containing microcentrifuge tube, and were difficult to dislodge. It is possible that many of the parasites were lost in this manner. In contrast, the drying procedure does not require field workers to transfer parasite samples from the dissecting slide. Furthermore, as the parasites are readily visible on the slide, they can be easily recovered by workers in the laboratory. An important question regarding the transmis- sion of 0. volvulus in West Africa is the relative vectorial capacity of sibling species in the S. damnosum s.l. complex. This has been difficult to address, since adult blackflies of the complex are hard to distinguish from one another. Recently, PCR-based methods of distinguishing members of the S. damnosum s.l. complex have been reported (8, 9). These methods rely on the PCR amplification of the internal transcribed spacer (ITS) of the rRNA gene cluster, and on the PCR amplification of a por- Table 1: Comparison of isopropanol-preserved and dried parasite samples in the 0-150 PCR assay No. No. No. Sample type assayed positive negative Isopropanol-preserved 153 73 80 microfilariae Dried microfilariae 272 210 62 Isopropanol-preserved 293 217 76 larvae Dried larvae 1019 998 21 tion of the NADH dehydrogenase subunit 4 (ND4) gene. To test the suitability of the dried malpighian tubule and ovary preparations for these assays, DNA was prepared from the dried blackfly material on approximately 200 of the slides. These samples were then used in separate PCR tests to amplify the ND4 and ITS gene sequences. The samples were found to be capable of reliably supporting PCR amplification of both the 238 bp ND4 fragment, as well as the 910 bp ITS fragment. Representative results are shown in Fig. 2. Fig. 2. Results of polymerase chain reaction amplifi- cation of the ITS (A) and ND4 (B) gene sequences from dried Simulium damnosum s.l. ovaries and malpigh- ian tubules. In each panel, M = 100bp ladder; 1 = ne- gative control; 2-9 = reactivity of DNA prepared from the field-collected samples. A?7 ITS M 1 2 3 4 5 6 7 8 9 B ....... WHO Bulletin OMS. Vol 75 1997 445 L. Toe et al. Thus, dried samples preserved on microscope slides can produce reliable substrates for PCR-based identification methods. The collection and preserva- tion method is simple, and samples produced by it are easily stored and transported. A further advan- tage is that both parasite and vector tissues pre- served in this way are suitable for PCR-based tests. This method should therefore be useful for studies of the relative vectorial capacity of different sibling blackfly species in the transmission of 0. volvulus under natural conditions. Acknowledgements We thank the Onchocerciasis Control Programme field teams for collecting the specimens analysed, and the cur- rent and former directors of the Onchocerciasis Control Programme, Dr Y. Dadzie and Dr E.M. Samba, for their support. This work was supported by grants from the Onchocerciasis Control Programme and the US National Institutes of Health. Resume Onchocerca volvulus: comparaison des methodes de collecte sur le terrain en vue de la conservation des specimens de parasites et de vecteurs destines a I'analyse par PCR Des methodes d'analyse moleculaire s'appuyant classiquement sur I'amplification et la d6tection de sequences specifiques de g6nome par PCR (ampli- fication enzymatique du genome) ont 6te mises au point ces dernieres annees pour l'identification d'Onchocerca volvulus et de son vecteur primaire, les simulies du complexe Simulium damnosum sensu lato (s.l.). Ces methodes posent toutefois certains problemes quant a la collecte des speci- mens sur le terrain. La m6thode id6ale de collecte des 6chantillons doit etre suffisamment simple et rapide pour pouvoir etre mise en ceuvre par des agents de terrain ayant la formation minimale. La m6thode doit en outre fournir des 6chantillons capables de supporter les conditions ambiantes difficiles de stockage et de transport, et ne doit donc pas n6cessiter des con- traintes de stockage et de manipulation qui im- poseraient des coOts suppl6mentaires. En fin de compte, la m6thode doit conserver l'echantillon dans un 6tat permettant le traitement au laboratoire, sans d6truire I'ADN qu'il contient et qui est necessaire pour la PCR. Les 6quipes de collecte sur le terrain et le laboratoire d'analyse par sondes d'ADN du Pro- gramme de lutte contre l'onchocercose en Afrique de l'Ouest ont proced6 pendant 2 ans a l'6valua- tion de deux methodes publi6es de collecte des 6chantillons publi6es qui paraissaient promet- teuses sur le terrain, et les ont comparees en tenant compte des criteres ci-dessus. La methode pref6r6e consiste a conserver le parasite et le materiel vectoriel s6lectionn6 a sec sur une lame, et est utilisable tant pour les parasites au stade microfilaire obtenus par biopsie cutanee exsangue chez I'homme, que pour les parasites au stade larve infectante obtenus sur les simulies. Une analyse a et6 realis6e sur plus de 1200 echantillons de parasites, et plus de 93% d'entre eux (1208/1291) avaient conserve assez d'ADN pour que le r6sultat avec la PCR 0-150, sp6cifique d'Onchocerca, soit positif. Le materiel vectoriel ainsi conserv6 a donn6 des r6sultats compar- ables. En conclusion, la conservation a sec, sur lame, des sp6cimens de parasite et de vecteur est une m6thode simple, bon marche, et efficace, de col-lecte sur le terrain des echantillons d'O. volvulus et du mat6riel vectoriel pour les analyses par PCR. References 1. Meredith SEO et al. 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