Detection of Brugia parasite DNA in human blood by real-time PCR

Abstract

Brugian filariasis (caused by the nematodes Brugia malayi and B. timori) is an important cause of disability in Southeast Asia. Improved diagnostic tests are needed for filariasis elimination programs (to identify areas of endemicity and to monitor progress) and for diagnosis of the disease in infected individuals. We have developed and evaluated two real-time PCR assays for detecting Brugia DNA in human blood and compared the results of these assays to those of "gold standard" assays. One assay uses a TaqMan probe (TaqM) to amplifiy a 320-bp "HhaI repeat" DNA sequence. The other assay uses a minor groove binding probe (MGB) and modified nucleotides in primers (Eclipse MGB) to amplify a 120-bp fragment of the HhaI repeat. This assay detects 22 copies of the target sequence, and it is more sensitive than the TaqM assay. Both assays were evaluated with human blood samples from two different areas of endemicity. The MGB assay was as sensitive as membrane filtration and microscopy for the detection of B. malayi infection in 57 blood samples recovered at night from patients in Sulawesi, Indonesia. The MGB assay also detected parasite DNA in 17 of 31 (55%) of microfilaria-negative day blood samples from these subjects. This test was more sensitive than the conventional and the TaqM PCRs (and was almost as sensitive as night blood membrane filtration) for the detection of infection in 52 blood samples recovered at night from individuals in an area of B. timori endemicity on Alor Island, Indonesia, where microfilaria-positive individuals had low densities after mass treatment. Thus, the Eclipse MGB real-time PCR assay is a sensitive means of detecting Brugia parasite DNA in human blood.

FIG. 1.

Sensitivities of the TaqM assay (gray lines) and Eclipse MGB (black lines) real-time PCR for detection of genomic Brugia malayi DNA (stars) or plasmid DNA (circles) containing the HhaI sequence. The amount of DNA (ng; x axis) is plotted against the cycle threshold values (y axis). Strong inverse correlations were observed between the amount of DNA and C_t values (both tests and both DNA samples; R values ranged from −0.9899 to −0.9964; P < 0.001).

FIG. 2.

Sensitivity of real-time PCR for detection of the HhaI sequence in blood samples. The numbers of copies of the HhaI repeat were calculated by using external standard curves. Copy numbers were plotted against blood microfilaria counts (mf/ml) on a double logarithmic scale. (A) TaqM assay results for Brugia malayi blood samples from Sulawesi (R = 0.767; P < 0.05); (B) TaqM assay results for Brugia timori blood samples from Alor Island (correlation not significant); (C) Eclipse MGB assay results for B. malayi blood samples from Sulawesi (R = 0.42; P < 0.05); (D) Eclipse MGB assay results for B. timori blood samples from Alor Island (correlation not significant).