BackgroundDiagnostic procedures for the diagnosis of infection with the nematode parasite Onchocerca volvulus are currently based on the microscopic detection of microfilariae in skin biopsies. Alternative approaches based on amplification of parasitic DNA in these skin biopsies are currently being explored. Mostly this is based on the detection of the O-150 repeat sequence using PCR based techniques.MethodsAn isothermal, loop-mediated amplification method has been designed using the mitochondrial O. volvulus cox1 gene as a target.ResultsAnalysis of dilution series of synthetic DNA containing the targeted sequence show a non-linear dose-response curve, as is usually the case for isothermal amplification methods. Evaluation of cross-reactivity with the heterologous sequence from the closely related parasites Wuchereria bancrofti, Loa loa and Brugia malayi demonstrated strong specificity, as none of these sequences was amplified. The assay however amplified both O. volvulus and O. ochengi DNA, but with a different melting point that can be used to discriminate between the species. Evaluation of this assay in a set of skin snip biopsies collected in an endemic area in Ghana showed a high correlation with O-150 qPCR and also demonstrated a similar sensitivity. Compared to qPCR, LAMP had a sensitivity of 88.2% and a specificity of 99.2%.ConclusionsWe have developed a sensitive and specific loop-mediated amplification method for detection of O. volvulus DNA in skin biopsies that is capable of providing results within 30 min.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1913-7) contains supplementary material, which is available to authorized users.

BackgroundOnchocerciasis, also known as river blindness is one of the neglected tropical diseases affecting millions of people, mainly in sub-Saharan Africa and is caused by the filarial nematode Onchocerca volvulus. Efforts to eliminate this disease are ongoing and are based on mass drug administration programs with the microfilaricide ivermectin. In order to monitor the efficacy of these programs, there is an unmet need for diagnostic tools capable of identifying infected patients. We have investigated the diagnostic potential of urinary N-acetyltyramine-O,β-glucuronide (NATOG), which is a promising O. volvulus specific biomarker previously identified by urine metabolome analysis.MethodsA liquid chromatography tandem mass spectrometry (LC-MS/MS) method was used to assess the stability characteristics of NATOG and to evaluate the levels of NATOG in study samples. An LC-fluorescence method was also developed.ResultsStability characteristics of NATOG were investigated and shown to be ideally suited for use in tropical settings. Also, an easy and more accessible method based on liquid chromatography coupled to fluorescence detection was developed and shown to have the necessary sensitivity (limit of quantification 1 μM). Furthermore, we have evaluated the levels of NATOG in a population of 98 nodule-positive individuals from Ghana with no or low levels of microfilaria in the skin and compared them with the levels observed in different control groups (endemic controls (n = 50), non-endemic controls (n = 18) and lymphatic filariasis (n = 51). Only a few (5 %) of nodule-positive individuals showed an increased level (> 10 μM) of NATOG and there was no statistical difference between the nodule-positive individuals and the control groups (P > 0.05).ConclusionsResults of the present study indicate the limited potential of NATOG as a diagnostic biomarker for O. volvulus infection in amicrofilaridermic individuals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1582-6) contains supplementary material, which is available to authorized users.

Abstract.Diagnostic tools for the detection of infection with Onchocerca volvulus are presently limited to microfilaria detection in skin biopsies and serological assessment using the Ov16 immunoglobulin G4 (IgG4) rapid test, both of which have limited sensitivity. We have investigated the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on immunodominant linear epitopes previously discovered. Peptides that were used in these assays were designated O. volvulus motif peptides (OvMP): OvMP-1 (VSV-EPVTTQET-VSV), OvMP-2 (VSV-KDGEDK-VSV), OvMP-3 (VSV-QTSNLD-VSV), and the combination of the latter two, OvMP-23 (VSV-KDGEDK-VSV-QTSNLD-VSV). Sensitivity (O. volvulus infection), specificity (non-helminth infections), and cross-reactivity (helminth infections) were determined using several panels of clinical plasma isolates. OvMP-1 was found to be very sensitive (100%) and specific (98.7%), but showed substantial cross-reactivity with other helminths. Of the other peptides, OvMP-23 was the most promising peptide with a sensitivity of 92.7%, a specificity of 100%, and a cross-reactivity of 6%. It was also demonstrated that these peptides were immunoreactive to IgG but not IgG4, and there is no correlation with the Ov16 IgG4 status, making them promising candidates to complement this already available test. Combination of the Ov16 IgG4 rapid test and OvMP-23 peptide ELISA led to a sensitivity of 97.3% for the detection of O. volvulus infection, without compromising specificity and with minimal impact on cross-reactivity. The available results open the opportunity for a “clinical utility use case” discussion for improved O. volvulus epidemiological mapping.