Smith ScholarWorks Biological Sciences: Faculty Publications Biological Sciences 1998 Detection of the Filarial Parasite Mansonella streptocerca in Skin Biopsies by a Nested Polymerase Chain Reaction-Based Assay Peter U. Fischer Dietrich W. Büttner Jotham Bamuhiiga Steven A. Williams Smith College, [email protected] Follow this and additional works at: https://scholarworks.smith.edu/bio_facpubs Part of the Biology Commons Recommended Citation Fischer, Peter U.; Büttner, Dietrich W.; Bamuhiiga, Jotham; and Williams, Steven A., "Detection of the Filarial Parasite Mansonella streptocerca in Skin Biopsies by a Nested Polymerase Chain Reaction-Based Assay" (1998). Biological Sciences: Faculty Publications, Smith College, Northampton, MA. https://scholarworks.smith.edu/bio_facpubs/41 This Article has been accepted for inclusion in Biological Sciences: Faculty Publications by an authorized administrator of Smith ScholarWorks. For more information, please contact [email protected] Am. J. Trop. Med. Hyg., 58(6), 1998, pp. 816±820 Copyright q 1998 by The American Society of Tropical Medicine and Hygiene DETECTION OF THE FILARIAL PARASITE MANSONELLA STREPTOCERCA IN SKIN BIOPSIES BY A NESTED POLYMERASE CHAIN REACTION±BASED ASSAY PETER FISCHER, DIETRICH W. BUÈ TTNER, JOTHAM BAMUHIIGA, AND STEVEN A. WILLIAMS Clark Science Center, Department of Biological Sciences, Smith College, Northampton, Massachusetts; Department of Helminthology and Entomology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; German Agency for Technical Cooperation and Basic Health Services, Fort Portal, Uganda Abstract. To differentiate the skin-dwelling ®lariae Mansonella streptocerca and Onchocerca volvulus, a nested polymerase chain reaction (PCR) assay was developed from small amounts of parasite material present in skin biopsies. One nonspeci®c and one speci®c pair of primers were used to amplify the 5S rDNA spacer region of M. streptocerca. Biopsies with different micro®laria densities obtained from 104 Ugandans living in an area endemic for M. streptocerca were tested using both the nested PCR assay and standard parasitologic assessment of micro®lariae. All 82 samples from micro®laria carriers were positive when tested using the nested PCR assay. In addition, M. streptocerca DNA could be detected in 16 samples thought to be micro®laria negative. Furthermore, six days follow- ing ivermectin treatment, M. streptocerca DNA was found in 12 of 14 micro®laria-negative biopsies. Control skin samples from patients infected with O. volvulus were all negative in the nested PCR assay. This assay improves the diagnosis of M. streptocerca and will facilitate further epidemiologic studies. When evaluating skin disease cases in large regions of MATERIALS AND METHODS Africa, the two skin-dwelling ®larial species, Mansonella streptocerca and Onchocerca volvulus, must be considered Patient samples. Skin biopsies were collected from per- as possible causative agents. Mansonella streptocerca infec- sons living in the Bundibugyo district located north of the tion is characterized by acute and more often chronic papular Ruwenzori mountains in western Uganda.2 This region is dermatitis mainly on the upper part of the body, which make endemic for M. streptocerca, but not for O. volvulus. For it dif®cult to differentiate streptocerciasis and onchocerciasis the evaluation of mf density, each biopsy was placed into a clinically.1, 2 In addition, after treatment with diethylcabar- single well of a microtiter plate containing phosphate buffer mazine or ivermectin, persons infected with M. streptocerca and was incubated overnight at room temperature. The so- also show a typical Mazzotti reaction.1, 3 Cross-reactions be- lution was than pipetted onto a glass slide for microscopic tween O. volvulus and Mansonella species impede the dif- examination using 633 magni®cation. Subsequently, the re- ferentiation of these parasites by serologic diagnosis using maining biopsy material from each well was preserved in worm extracts.4 Although isolated micro®lariae (mf) of both 80% ethanol and stored in a refrigerator for later PCR test- species can be differentiated morphologically, species dif- ing. For comparison, skin snips were collected from patients ferentiation in histologic sections is dif®cult. Since the mf living in an area of western Uganda endemic for O. volvulus, densities of M. streptocerca are often relatively low, meth- but not for M. streptocerca.12 Genomic O. volvulus DNA ods other than the standard skin snip technique are required was also prepared from female worms isolated from extir- for accurate detection of this species. Collagenase digestion pated nodules from patients in the same area. In both the M. of the skin snips is an appropriate procedure,2 but if a patient streptocerca- and O. volvulus-endemic regions, the blood ®- is also heavily infected with O. volvulus it may be dif®cult larial parasite Mansonella perstans was coendemic. No other to detect one M. streptocerca mf among hundreds of O. vol- ®larial species infecting humans were found in these vulus mf. In these cases, a speci®c but more sensitive assay regions.2, 13 Single specimens from ®lariasis patients of other to detect M. streptocerca is clearly required. geographic regions were also available from previous stud- The polymerase chain reaction (PCR) is a sensitive tech- ies. Informed consent was obtained from all individuals who nique to detect parasite DNA. Sensitive and speci®c PCR participated in the investigation. The study was approved by assays have been developed for the diagnosis of the human the Ministry of Health of Uganda and by the Ethics Com- ®larial parasites Wuchereria bancrofti, Brugia malayi, Loa mission of the Medical Board of Hamburg, Germany. loa, and O. volvulus.5±10 All of these assays are based on All persons were examined for ®larial skin lesions before different repetitive target sequences found only within a dis- and six days following ivermectin treatment according to the tinct ®larial genus. In the present study, we have developed recommendations by Murdoch and others14 and the World a PCR assay based on a target sequence present in all ®larial Health Organization.15 The skin reaction of micro®laria car- and all other nematode species.11 The 5S rDNA spacer re- riers following administration of micro®laricidal drugs gion was ampli®ed from DNA prepared from human skin (Mazzotti reaction) was used as an additional tool to deter- biopsies that contained only a small number of M. strepto- mine infection. Only fresh papules seen by the examiner cerca mf. The primers used in the assay are based on con- following treatment were recorded as a positive Mazzotti test served coding regions of the 5S rDNA. After sequencing of result. Most patients presented a history of increased itching the 5S rDNA spacer PCR product, species-speci®c primers following treatment. Since such a statement cannot be veri- were designed to detect M. streptocerca speci®cally in a ®ed by the examiner, it was not used as a criterion for a nested PCR assay. The sensitivity and speci®city of this as- positive Mazzotti test result. All consenting persons were say was evaluated on clinical samples collected in an en- treated with a single oral dose of ivermectin (150 mg/kg of demic area in western Uganda. body weight), except three who presented with pregnancy, 816 DETECTION OF M. STREPTOCERCA USING PCR 817 FIGURE 1. Sequence of the 5S rDNA spacer region, the location of the second set of primers (MsU and MsL), and the location of the spliced leader sequence in Mansonella streptocerca, M. perstans, and Onchocerca volvulus. asthma, and a doubtful neurologic disorder. A few patients bromide/UV illumination. For the second PCR, the same cy- did not come for the follow-up examination and some were cling conditions were chosen except that only 1 ml of the only treated at the second visit. Therefore, 73 patients were PCR product was used as template and only 25 cycles were re-examined for a Mazzotti reaction. performed. The primers used in the second PCR are shown Sample preparation and PCR. The DNA was prepared in Figure 1. from skin snips, micro®lariae, and adult O. volvulus as de- Sequencing of DNA and nested PCR primer design. scribed previously.16 For some samples, a simpli®ed sample Dideoxy sequencing of PCR products was performed by di- preparation method was used. Fifty microliters of nucleoly- rect cycle sequencing using one of the PCR primers as a sis buffer (0.05 M NaCl, 0.01 M EDTA, 0.05 M Tris base) sequencing primer. First, the PCR products were puri®ed us- and 25 ml of 1 N NaOH were added to the samples and ing silica-gel membrane spin-columns (Qiaquick PCR Puri- incubated for 1 hr at 608C. The solution was then neutralized ®cation Kit; Qiagen, Hilden, Germany). Fluorescence-based using 1 N HCl and the DNA was ethanol precipitated over- sequencing was performed using the ABI Prism DNA Se- night at 2208C. The DNA was pelleted by centrifuging at quencing Kit following the protocol of the manufacturer 13,800 3 g for 20 min, dried and resuspended in 50 mlof (Applied Biosystems Division, Perkin Elmer, Foster City, Tris-EDTA buffer (0.5 M Tris base, 0.05 M EDTA). Two CA). Unincorporated dideoxy terminators were removed us- microliters of each extraction were subjected to the ®rst ing Centri-Sep columns (Princeton Separation, Adelphia, PCR. During the DNA preparation and the PCR, standard NJ). Subsequently, the sequencing reactions were run on an precautions were applied to prevent contamination. Skin automated sequencer (ABI 373A; Perkin Elmer). Results snips from areas nonendemic for M. streptocerca functioned were further analyzed using the programs Seq Ed version as negative controls during the DNA preparation procedure 1.0.3 (ABD; Perkin Elmer) and Oligo 5.0 (National Biosci- and all mf negative samples were tested twice in the nested ence Inc., Plymouth, MN) run on Macintosh (Cupertino, PCR assay. Unless speci®cally noted, all chemicals were ob- CA) Power PC computers.