Rapid Molecular Assays for Specific Detection and Quantitation of Loa loa Microfilaremia

Abstract

BackgroundAccurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.Methodology/Principal FindingsFrom a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.Conclusions/SignificanceThis novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.

Loop-Mediated Isothermal Amplification for Rapid and Semiquantitative Detection of Loa loa Infection

“…The source of genomic DNA from L. loa, W. bancrofti, Brugia malayi, and Mansonella perstans microfilariae (mf) has been described previously (26). For some experiments, 100,000 purified L. loa mf were placed in a fixed volume (1 ml) of distilled water and serially diluted to create standards for quantification.…”

“…Alternative serological (22,23) and molecular (24)(25)(26) tests have been developed, but to date, only real-time PCR methodology has been able to combine a high degree of sensitivity and specificity with the ability to accurately quantify L. loa mf levels (26,27). These methods, however, require a centralized, wellequipped laboratory and relatively expensive reagents.…”

“…In the present study, we have used two L. loa-specific singlecopy target genes, LLMF72 and LLMF342, to design LAMP primers for detection of L. loa mf in human blood and have developed methods that have a sensitivity and specificity similar to those of the previously described quantitative PCR (qPCR) assay targeting the LLMF72 gene (26). Furthermore, we have identified LAMP conditions that allow for colorimetric determination of microfilarial levels above or below specified thresholds such that our LAMP method now has the potential to be a practical point-of-care tool for identifying people at risk for SAEs when given IVM.…”

Discovery of Specific Antigens That Can Predict Microfilarial Intensity in Loa loa Infection

“…These North American volunteers (normal controls) had no history of exposure to filariae or other helminths, had never traveled outside North America, and were collected through a healthy volunteer research protocol from the Division of Transfusion Medicine at the National Institutes of Health. The diagnosis of mf ؉ L. loa and W. bancrofti was made based on the presence of mf by microscopy in a blood smear (and/or in a filtered 1-ml blood sample) or by PCR (11,29). The presence of O. volvulus mf was determined by skin snip examination by microscopy.…”

“…Real-time quantitative PCR (qPCR) and loop-mediated isothermal amplification (LAMP) methods are credible alternatives to microscopy since they are high throughput and combine a high degree of sensitivity and specificity with the ability to accurately quantify L. loa mf levels (9)(10)(11). However, they require a well-equipped laboratory (for qPCR), relatively expensive reagents, and time-intensive DNA/RNA extraction processes.…”