Real-time PCR detection of the HhaI tandem DNA repeat in pre- and post-patent Brugia malayi infections: a study in Indonesian transmigrants

Abstract

BackgroundLymphatic filariasis caused by Wuchereria bancrofti or Brugia spp. is a public health problem in developing countries. To monitor bancroftian filariasis infections, Circulating Filarial Antigen (CFA) test is commonly used, but for brugian infections only microfilariae (Mf) microscopy and indirect IgG4 antibody analyses are available. Improved diagnostics for detecting latent infections are required.MethodsAn optimized real-time PCR targeting the brugian HhaI repeat was validated with plasma from microfilariae negative Mongolian gerbils (jirds) infected with B. malayi. Plasma samples from microfilaremic patients infected with B. malayi or W. bancrofti were used as positive and negative controls, respectively. PCR results of plasma samples from a transmigrant population in a B. malayi endemic area were compared to those of life-long residents in the same endemic area; and to IgG4 serology results from the same population. To discriminate between active infections and larval exposure a threshold was determined by correlation and Receiver-Operating Characteristics (ROC) curve analyses.ResultsThe PCR detected HhaI in pre-patent (56 dpi) B. malayi infected jirds and B. malayi Mf-positive patients from Central Sulawesi, Indonesia. HhaI was also detected in 9/9 elephantiasis patients. In South Sulawesi 87.4% of the transmigrants and life-long residents (94% Mf-negative) were HhaI PCR positive. Based on ROC-curve analysis a threshold for active infections was set to >53 HhaI copies/μl (AUC: 0.854).ConclusionsThe results demonstrate that the HhaI PCR detects brugian infections with greater sensitivity than the IgG4 test, most notably in Mf-negative patients (i.e. pre-patent or latent infections).

Diagnostic and prognostic value of cell free circulating Schistosoma mansoni DNA: an experimental study

“…in an experimental model (Sandoval et al 2006b), suggesting the possibility of excretion of free circulating pathogen DNA in urine. Similarly, other parasites, free circulating DNA was detected by PCR during early infection periods in the experimental models for Trypanosoma cruzi (Kirchhoff et al 1996), filariasis (Albers et al 2014) and Toxoplasma gondii (Weiss et al 1991;Wang et al 2012), thus allowing the diagnosis of infection earlier than when using conventional diagnostic techniques as microscopy or serology. The dynamic of free DNA level during the next samples showed continuous increase in the DNA level throughout the infection progression (until the beginning of treatment 30 days p.i.)…”

Expanding the MDx toolbox for filarial diagnosis and surveillance

“…High copy number and nucleotide identity of HhaI repeats have engendered high levels of sensitivity in various PCR-related platforms [10,34]. Recently, a real-time PCR assay targeting the HhaI repeat was demonstrated to be significantly more sensitive than antibody tests for detection of pre-patent and amicrofilaremic patients using day blood samples [10].…”

“…High copy number and nucleotide identity of HhaI repeats have engendered high levels of sensitivity in various PCR-related platforms [10,34]. Recently, a real-time PCR assay targeting the HhaI repeat was demonstrated to be significantly more sensitive than antibody tests for detection of pre-patent and amicrofilaremic patients using day blood samples [10]. Other targets described for B. malayi diagnosis include an L3-activated collagen transcript [35], the internal transcribed spacer 1 region of rDNA [36], glutathione peroxidase [37], and intron-3 tandem repeats of alt-2 [38].…”

“…These activities and overall management of such control programs are most efficiently performed with accurate diagnostic tools suitable for field use. Highly-sensitive methods are required to detect low mf densities in skin or blood, which is often the case when MDA programs are underway [8,9], as well as occult and amicrofilaremic infections [10]. Stringent specificity is required to differentiate the closely related filarial parasites, especially in the equatorial rain forest areas of central and western Africa where loiasis is endemic, because administration of ivermectin can lead to encephalopathy in patients coinfected with L. loa.…”

Technetium-99m-Labeled Diethylcarbamazine Citrate (^99mTc-DEC) as a New Diagnostic Agent for Lymphatic Filariasis Detection

“…Therefore, early detection for this case is quite difficult to attain. Laboratory tests for identification of filarial antigens by enzyme-linked immunosorbent assay (ELISA) technique or rapid immunochromatographic card can actually be done, but those techniques are complicated and often give false positive results [5][6][7][8][9][10][11][12]. Diagnosis techniques with higher sensitivity and specificity are still needed .…”