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.

Molecular Diagnostics in the Diagnosis of Parasitic Infection

“…Molecular assays, including conventional and real-time PCR formats and LAMPbased methods, have been described for the four major filarial pathogens (Drame, Fink, Kamgno, Herrick, & Nutman, 2014;Fischer, Supali, Wibowo, Bonow, & Williams, 2000;Hassan et al, 2005;Kluber, Supali, Williams, Liebau, & Fischer, 2001;Lizotte, Supali, Partono, & Williams, 1994;McCarthy et al, 1996;Ramzy, 2002;Rao, Atkinson, et al, 2006;Rao, Weil, Fischer, Supali, & Fischer, 2006;Takagi et al, 2011;Toe et al, 1998;Toure et al, 1997Toure et al, , 1998Zhong et al, 1996;Zimmerman et al, 1994). Common targets are the 188 bp DNA non-coding DNA repeat sequence Ssp-I for W. bancrofti (Ramzy, 2002;Rao, Atkinson, et al, 2006;Takagi et al, 2011;Zhong et al, 1996), Hha I repeat region for B. malayi (Fischer et al, 2000;Lizotte et al, 1994;Rao, Weil et al, 2006), LLMF72 or a repeat region (15r3) for L. loa (Drame et al, 2014;Fink, Kamgno, & Nutman, 2011;Toure et al, 1998), and O-150 repeat sequence for O. volvulus (Toe et al, 1998;Zimmerman et al, 1994).…”

“…Common targets are the 188 bp DNA non-coding DNA repeat sequence Ssp-I for W. bancrofti (Ramzy, 2002;Rao, Atkinson, et al, 2006;Takagi et al, 2011;Zhong et al, 1996), Hha I repeat region for B. malayi (Fischer et al, 2000;Lizotte et al, 1994;Rao, Weil et al, 2006), LLMF72 or a repeat region (15r3) for L. loa (Drame et al, 2014;Fink, Kamgno, & Nutman, 2011;Toure et al, 1998), and O-150 repeat sequence for O. volvulus (Toe et al, 1998;Zimmerman et al, 1994).…”

Arbonematodes – Nematode Infections Transmissible by Arthropods

“…Both detection methods were negative for B. pahangi (occurring in monkeys), and W. bancrofti microfilariae [44]. A one-step multiplex PCR for the simultaneous detection of B. malayi and W. bancrofti Ssp I repeat DNA was developed by Mishra et al [45].…”

Differential detection of Brugia malayi and Brugia pahangi by real-time fluorescence resonance energy transfer PCR and its evaluation for diagnosis of B. pahangi-infected dogs

“…Brugian filariasis caused by Brugia malayi and Brugia timori is a mosquito-borne disease that affects approximately 13 million people, mainly in India and Southeast Asia (Rao et al 2006). Possible evidence of natural infection of man with Brugia pahangi in South Kalimantan (Borneo), Indonesia was reported (Palmieri et al 1985).…”