Evaluation of a Polymerase Chain Reaction-Based Assay for Diagnosis of Wuchereria bancrofti Infection

The American Journal of Tropical Medicine and Hygiene

Büttner²,

Bamuhiiga³

et al. 1998

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...

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confidence: 99%

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

The American Journal of Tropical Medicine and Hygiene

Büttner²,

Bamuhiiga³

et al. 1998

“…Therefore, this assay has at least the same or even a higher sensitivity than conventional PCR assays for the detection of filarial parasites. 7,[9][10][11][12] It has been reported that a conventional PCR-ELISA without any internal control is a very sensitive tool for the detection of PCR products of the B. malayi Hha I repeat. 13 Our results confirmed this finding using a different labeling hapten and a different antibodyconjugate for the ELISA detection.…”

Section: Discussionmentioning

confidence: 99%

“…4,5 Various sensitive polymerase chain reaction (PCR) assays have been established to detect W. bancrofti or B. malayi DNA in the human host or the mosquito vector. [6][7][8][9][10][11][12][13][14][15] Inhibition of a PCR, which may occur in both samples obtained from the mosquito vector and samples from the human host, is a common problem during DNA amplification and makes the standardization of PCR tests difficult. 16 Therefore, DNA constructs were used as internal standards (controls) in the PCR to avoid false-negative results in a diagnostic PCR.…”

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confidence: 99%

Detection of DNA of nocturnally periodic Brugia malayi in night and day blood samples by a polymerase chain reaction-ELISA-based method using an internal control DNA.

The American Journal of Tropical Medicine and Hygiene

Supali²,

Wibowo³

et al. 2000

Abstract. An internal control was used in a polymerase chain reaction (PCR)-ELISA-based technique to detect the Hha I repeat of the filarial parasite Brugia malayi. A single microfilaria added to 200 l of blood was reliably detected. The assay was evaluated on field samples from persons living in an area endemic for Anopheles-transmitted, nocturnally periodic B. malayi in central Sulawesi, Indonesia. Examination of night blood of 138 individuals for the presence of microfilariae by filtration revealed 44 microfilaria carriers. All microfilaria carriers were also positive in the PCR-ELISA and, in addition, 14 more samples were proven to contain parasite DNA. The sensitivity of both methods was compared on night and on day blood samples collected from 113 persons. Whereas 37 microfilaria carriers were identified by filtration of night blood, no microfilariae were observed in the corresponding day blood samples. The PCR-ELISA result was positive in all 37 night blood samples of microfilaria carriers and in an additional 13 night blood samples without microfilariae. Parasite DNA was detected in 31 day blood samples of microfilaria carriers and in 3 day blood samples of amicrofilaremic persons. Assuming a sensitivity of the PCR-ELISA on night blood of 100%, the sensitivity of night blood filtration is 74% and that of the PCR-ELISA on day blood is 68%. These data suggest that the described PCR-ELISA method is capable of detecting infections with nocturnally periodic B. malayi in day blood samples. Therefore, this method may facilitate both the identification of endemic areas and the monitoring of control programs.Lymphatic filariasis, caused by Wuchereria bancrofti and Brugia malayi, has been targeted by the World Health Organization for elimination as a public health problem by the year 2020. 1 Therefore, control programs have been or will be launched world-wide with an emphasis on communitywide treatment with diethylcarbamazine (DEC) and ivermectin or albendazole.2 To monitor the success of such intervention programs, sensitive and cost effective diagnostic tools are required. It is estimated that 115 million people are infected with W. bancrofti and about 13 million with B. malayi. 3 In Asia, 1 of 5 filarial infections is due to B. malayi and in some countries this parasite is responsible for the majority of infections. 4,5

“…[7][8][9][10][11] Screening of pools of mosquitoes would also speed up the processing of large numbers of specimens. A PCR assay for pool screening targeting a repeated DNA segment (Ssp I repeat) in the W. bancrofti genome has been successfully applied to culicine mosquitoes including Aedes polynesiensis 8 and Culex quinquifasciatus.…”

Section: Introductionmentioning

confidence: 99%

Application of a polymerase chain reaction-ELISA to detect Wuchereria bancrofti in pools of wild-caught Anopheles punctulatus in a filariasis control area in Papua New Guinea.

Bockarie

The American Journal of Tropical Medicine and Hygiene

Williams

et al. 2000

Abstract. Chemotherapy-based eradication programs are aimed at stopping transmission of Wuchereria bancrofti by its obligatory mosquito vector. This study compares one year post-treatment W. bancrofti infection rates of Anopheles punctulatus, the main vector of lymphatic filariasis in Papua New Guinea, using traditional dissection techniques and a polymerase chain reaction (PCR)-based ELISA of a parasite-specific Ssp I repeat. A total of 633 mosquitoes in 35 batches were dissected. Six batches contained W. bancrofti-infected mosquitoes, giving a minimum infection rate of 0.9%. This value was not different than the actual infection rate, which was 9 (1.4%) of 633 mosquitoes (P ϭ 0.48). The DNA was extracted from 47 pools containing a mean of 13.2 mosquitoes per pool. A total of 621 mosquitoes were processed for the PCR-ELISA, including 486 caught by human bait and 135 by light trap, which included both dead and live mosquitoes. Of 23 pools of alcohol-preserved human-bait mosquitoes, seven were positive by the PCR-ELISA, giving an infection rate identical to that obtained by dissection of individual mosquitoes (1.4%). The minimum infection rates for pools of light-trap mosquitoes found dead and alive were 2.7% (2 of 74) and 4.9% (3 of 61), respectively. These values did not differ from each other (P ϭ 0.84), but the overall infection rate of lighttrap mosquitoes was greater than that of mosquitoes captured by human bait (3.7% versus 1.4%; P ϭ 0.09). These data indicate that the PCR-ELISA of a W. bancrofti Ssp I repeat using pools of mosquitoes is comparable to traditional dissection techniques for monitoring transmission intensity following introduction of mass chemotherapy. This approach may also be useful for rapid and cost-effective assessment of transmission in endemic areas where the frequency of overt lymphatic pathology is low.