A TaqMan-based multiplex real-time PCR assay for the simultaneous detection of Wuchereria bancrofti and Brugia malayi

Pilotte, Nils; Torres, Melissa; Tomaino, Francesca Rachael; Laney, Sandra J.; Williams, Steven A.

doi:10.1016/j.molbiopara.2013.05.001

Cited by 22 publications

(20 citation statements)

References 11 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Thiele¹,

Cama²,

Lakwo³

et al. 2016

The American Society of Tropical Medicine and Hygiene

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Abstract. Microscopic evaluation of skin biopsies is the monitoring and evaluation (M and E) method currently used by multiple onchocerciasis elimination programs in Africa. However, as repeated mass drug administration suppresses microfilarial loads, the sensitivity and programmatic utility of skin snip microscopy is expected to decrease. Using a pan-filarial real-time polymerase chain reaction with melt curve analysis (qPCR-MCA), we evaluated 1) the use of a single-step molecular assay for detecting and identifying Onchocerca volvulus microfilariae in residual skin snips and 2) the sensitivity of skin snip microscopy relative to qPCR-MCA. Skin snips were collected and examined with routine microscopy in hyperendemic regions of Uganda and Ethiopia (N = 500 each) and "residual" skin snips (tissue remaining after induced microfilarial emergence) were tested with qPCR-MCA. qPCR-MCA detected Onchocerca DNA in 223 residual snips: 139 of 147 microscopy(+) and 84 among microscopy(−) snips, suggesting overall sensitivity of microscopy was 62.3% (139/223) relative to qPCR-MCA (75.6% in Uganda and 28.6% in Ethiopia). These findings demonstrate the insufficient sensitivity of skin snip microscopy for reliable programmatic monitoring. Molecular tools such as qPCR-MCA can augment sensitivity and provide diagnostic confirmation of skin biopsies and will be useful for evaluation or validation of new onchocerciasis M and E tools.

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Section: Discussionmentioning

confidence: 99%

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

Thiele¹,

Cama²,

Lakwo³

et al. 2016

The American Society of Tropical Medicine and Hygiene

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“…The 320-bp HhaI tandem repeat sequence of B. malayi has been used as a target for B. malayi PCR assays for many years [19,20]. Rao et al .…”

Section: Discussionmentioning

confidence: 99%

“…Using a real-time PCR assay targeting the B. malayi HhaI repeat, a sensitive and specific detection of nocturnally periodic B. malayi DNA in day blood samples was possible including 33% of Mf-negative samples in a highly endemic area [19]. Recently a multiplex real-time PCR assay for the B. malayi HhaI and W. bancrofti Long DNA Repeat was established for use in co-endemic areas [20]. This once more showed that real-time PCR assays provide a significant cost and labor saving procedure for disease monitoring efforts in endemic locations as part of the Global Progamme for the Elimination of Lymphatic Filariasis (GPELF) [20].…”

Section: Introductionmentioning

confidence: 99%

“…Recently a multiplex real-time PCR assay for the B. malayi HhaI and W. bancrofti Long DNA Repeat was established for use in co-endemic areas [20]. This once more showed that real-time PCR assays provide a significant cost and labor saving procedure for disease monitoring efforts in endemic locations as part of the Global Progamme for the Elimination of Lymphatic Filariasis (GPELF) [20]. …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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).

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“…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].…”

Section: Reviewmentioning

confidence: 99%

Expanding the MDx toolbox for filarial diagnosis and surveillance

Alhassan¹,

Li²,

Poole³

et al. 2015

Trends in Parasitology

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A TaqMan-based multiplex real-time PCR assay for the simultaneous detection of Wuchereria bancrofti and Brugia malayi

Cited by 22 publications

References 11 publications

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

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

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

Expanding the MDx toolbox for filarial diagnosis and surveillance

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