In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi

Abstract

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans , M . ozzardi , M . rodhaini and M . streptocerca . Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M . perstans and M . ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M . perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000 th of a microfilaria, while the M . ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M . perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

Aplicación de la técnica LAMP para la detección de Loa loa y Mansonella perstans

“…Para solventar estos problemas, la tecnología LAMP es una excelente alternativa a la PCR (Mori y Notomi, 2020). En este trabajo utilizamos el LAMP para detectar ADN de Loa loa (Fernández-Soto et al, 2014) y ADN de M. perstans (Poole et al, 2019) evaluando su eficacia en el diagnóstico de 22 muestras clínicas seleccionadas de un estudio realizado en Guinea Ecuatorial. El LAMP de Loa loa resultó positivo en las 5 muestras confirmadas parasitológicamente, coincidiendo con los resultados de qPCR (n. os 16-20).…”

“…Este LAMP solo ha sido evaluado con muestras artificiales preparadas en el laboratorio. Sería de gran interés evaluar su funcionamiento en muestras clínicas de individuos residentes en zona endémica de loaosis y también evaluar un LAMP para la detección de M. perstans (Poole et al, 2019), pues su diagnóstico pasa frecuentemente inadvertido.…”

“…El LAMP para la detección de Loa loa se realizó utilizando el método de Fernández-Soto et al (2014) y para M. perstans, según lo descrito por Poole et al (2019). Las reacciones se realizaron en un termobloque a 60 ºC durante 45 min, más 10 min a 80 ºC para inactivar la enzima.…”

Multiple origins of nematode-Wolbachiasymbiosis in supergroup F and convergent loss of bacterioferritin in filarialWolbachia

“…DNA was extracted using a Genomic DNA Tissue MicroPrep kit (Zymo Research, USA) as described previously 37 . DNA from Mpe2 was isolated as described above for Moz2.…”

“…The NEBNext Microbiome DNA enrichment kit (New England Biolabs Inc., USA) was used as directed to enrich for parasite DNA and reduce human DNA contamination prior to construction of the libraries from each of the samples, except Moz2. The preparation of Illumina libraries from Mpe1 and Moz1 samples have been described as part of a previous study 37 . A similar protocol was used for preparing libraries from Mpe2 and Moz2.…”