Detection of the filarial parasite Mansonella streptocerca in skin biopsies by a nested polymerase chain reaction-based assay.

Abstract

Abstract. To differentiate the skin-dwelling filariae 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 nonspecific and one specific pair of primers were used to amplify the 5S rDNA spacer region of M. streptocerca. Biopsies with different microfilaria 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 microfilariae. All 82 samples from microfilaria carriers were positive when tested using the nested PCR assay. In addition, M. streptocerca DNA could be detected in 16 samples thought to be microfilaria negative. Furthermore, six days following ivermectin treatment, M. streptocerca DNA was found in 12 of 14 microfilaria-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 Africa, the two skin-dwelling filarial species, Mansonella streptocerca and Onchocerca volvulus, must be considered as possible causative agents. Mansonella streptocerca infection is characterized by acute and more often chronic papular dermatitis mainly on the upper part of the body, which make it difficult to differentiate streptocerciasis and onchocerciasis clinically. 1, 2 In addition, after treatment with diethylcabarmazine or ivermectin, persons infected with M. streptocerca also show a typical Mazzotti reaction. 1, 3 Cross-reactions between O. volvulus and Mansonella species impede the differentiation of these parasites by serologic diagnosis using worm extracts. 4 Although isolated microfilariae (mf) of both species can be differentiated morphologically, species differentiation in histologic sections is difficult. Since the mf densities of M. streptocerca are often relatively low, methods other than the standard skin snip technique are required for accurate detection of this species. Collagenase digestion of the skin snips is an appropriate procedure, 2 but if a patient is also heavily infected with O. volvulus it may be difficult to detect one M. streptocerca mf among hundreds of O. volvulus mf. In these cases, a specific but more sensitive assay to detect M. streptocerca is clearly required.The polymerase chain reaction (PCR) is a sensitive technique to detect parasite DNA. Sensitive and specific PCR assays have been developed for the diagnosis of the human filarial parasites Wuchereria bancrofti, Brugia malayi, Loa loa, and O. volvulus. 5-10 All of these assays are based on different repetitive target sequences found only within a distinct filarial genus. In the present study, we have developed a PCR assay based on a target sequence present in all filarial and all other nematode species. 11 The 5S rDNA spacer region was amplified from DNA prepared from human skin biopsi...

Genetic Characterization of Atypical Mansonella (Mansonella) ozzardi Microfilariae in Human Blood Samples from Northeastern Peru

“…For example, a nested PCR-based assay easily differentiated between M. (E.) streptocerca and O. volvulus, which are difficult to distinguish by morphological examination. 16 Our results show that the atypical Mf from Peru are identical or very closely related to M. ozzardi. Minor morphological differences described in preliminary reports may reflect natural variation within the species 5 or can be artificially seen from stained fixed Mf in slides during the field study because of irregular contractures of the Mf body that can also modify the length as described above.…”

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities

“…methods include the O-150 PCR-ELISA used here, qPCR-MCA of the O-150 repeat, TaqMan-based qPCR, loopmediated isothermal amplification, specific oligonucleotide capture with magnetic beads, nested PCR combining general and species-specific primers, and PCR with general filarial primers followed by restriction fragment length polymorphism analysis to determine taxonomic identity. 13,[15][16][17][18][19][20][21][22][23] However, with few exceptions, these procedures require at least one post-amplification step for final determination, are specific to only one genus, or require multiple specific reaction components. 16,17 The procedure used here allows for singletube assessment of both presence and identity in a single 90-minute reaction.…”

The morphological discrimination of microfilariae of Onchocerca volvulus from Mansonella ozzardi

“…Hence there is a real risk of misidentification of M. ozzardi during clinical examination of patients for the better known skin-dwelling microfilariae such as Onchocerca volvulus (Leuckart, 1893). This sort of problem led to the initial misidentification of M. streptocerca for O. volvulus in Uganda where M. streptocerca was not expected (Fischer et al 1998). Mansonella ozzardi and O. volvulus are only known to be sympatric in the Amazonian focus of onchocerciasis which straddles the Brazil-Venezuela frontier ( Fig.…”