Small non-coding RNAs and Filarial infections

What are Neglected Tropical Diseases (NTDs)?

The World Health Organization (WHO) defines NTDs as a group of communicable, poverty-promoting diseases, which affect more than one billion people worldwide (Herricks et al., 2017). These diseases are primarily concentrated in sub-Saharan Africa, as well as in regions of South America and Asia (Herricks et al., 2017). The stigma surrounding these diseases, and their impact on children and women’s health, and worker productivity, are all factors that negatively contribute to the economic burden in these countries (Turner et al., 2016; Herricks et al., 2017). Traditionally, NTDs have carried a low priority on donor agendas when compared to other diseases such as HIV/AIDS and malaria, thus accounting for the term “neglected” (Turner et al., 2016; Herricks et al., 2017). At present, a total of 17 bacterial and parasitic infections are catalogued as NTDs, and among others, include dengue, trypanosomiasis, ascariasis, leprosy, schistosomiasis, and filarial infections (Herricks et al., 2017).

What are filarial infections?

Filarial infections are a group of chronic and extremely debilitating NTDs affecting more than 100 million people in Africa and South America (Taylor, Hoerauf and Bockarie, 2010; Ballesteros et al., 2016). Depending upon the parasite species, these diseases can be broadly classified into lymphatic filariasis, also known as elephantiasis (caused by Wuchereria bancrofti and Brugia malayi), and onchocerciasis, also known as river blindness (Onchocerca volvulus) [2]. These parasites are transmitted to humans by either mosquitos (lymphatic filariasis) or blackflies (onchocerciasis) [2]. The pathology associated with lymphatic filariasis is characterised by a concomitant malformation of the lower limbs and groin, inducing a long-lasting impairment in mobility, whereas onchocerciasis is characterised by visual deterioration and blindness [2]. These diseases impose a tremendous social and economic burden on endemic regions, due not only to the stigma surrounding them, but also due to the incapacity of patients to have a normal life, thus perpetuating a cycle of poverty.

What are the current challenges in the diagnostics of filarial infections?

At present, several programmes carried out by the WHO aim to control transmission and eradicate these diseases from endemic foci in African and South American countries. To achieve this ambitious goal, health workers and medical personal need to be able to identify and treat infected patients easily and effectively. Currently, the gold-standard diagnostic tool involves the detection of the transmission stage of the parasite, termed microfilariae (mf), in blood samples or skin biopsies (Weil and Ramzy, 2007; Rebollo and Bockarie, 2017). Mf are released, in their thousands, by adult female worms residing within infected patients (found within the lymphatics or in subcutaneous nodules), and are normally found in the bloodstream (lymphatic filariasis) or in the skin (onchocerciasis). However, this diagnostic method is labour-intensive and causes considerable pain and discomfort. Moreover, treatment with the chemotherapeutic agent Ivermectin reduces the mf output by gravid adult female worms, further compromising the efficacy of mf identification as a biomarker to diagnose filariasis patients; not to mention its poor performance for treatment monitoring purposes (Guzmán et al., 2002; Eberhard et al., 2017).

Nucleic acid-based diagnostic tools for filarial infections

Apart from immunoassays for the detection of parasite antigens such as the O. volvulus antigen Ov16 (Park, Dickerson and Janda, 2008), and the Brugia spp. antigens BmR1 and Bm14 (Rahmah et al., 2001), other diagnostic tools involve the detection of parasite-derived nucleic acids in biofluids. In this context, the Brugia DNA repetitive element HhaI has been detected in blood by Loop-mediated Isothermal Amplification (LAMP), and has been demonstrated to be a rapid method that is highly sensitive and specific to Brugian infections (Poole et al., 2012). Apart from DNA-based tests, the detection of other nucleic acids, such as small non-coding RNAs has also become popular as attractive biomarkers for filarial infections (Quintana, Babayan and Buck, 2016; Tritten and Geary, 2016).

Extracellular parasite-derived microRNAs as biomarkers for filarial infections

MicroRNAs (miRNAs) are ~21-24 nt long small non-coding RNAs with important regulatory functions controlling gene expression in the cell. First described in Caenorhabditis elegans over two decades ago, miRNAs are encoded within the genome as stem-loop structures that undergo a series of maturation events to produce the short RNA guide. This short guide RNA is normally found within the cell in association with an RNA-binding protein of the Argonaute (AGO) family, forming the RNA-Induced Silencing Complex (RISC), which recognises its cognate messenger RNA target by base complementarity. However, it is now recognized that RNA molecules can also operate beyond the limits of the cell. One key feature of extracellular parasite-derived RNAs (exRNA) is its remarkable stability in hostile environments such as biofluids (e.g. serum, plasma, urine, tears, saliva, among others). Several studies have demonstrated that the stabilization of exRNA can occur through direct association with protein and lipid partners such as AGO complexes or High-density lipoprotein (HDL) particles or encapsulation within extracellular vesicles (EVs) (Reviewed in (Quintana, Babayan and Buck, 2016)).

One potential application of these parasite-derived exRNAs is as biomarkers for parasitic infections. This is based on a key observation that parasite-derived exRNAs can be detected in biofluids from their hosts as demonstrated by small RNA sequencing and qRT-PCR (Figure 1) (Tritten et al., 2014; Quintana et al., 2015). We, and others, have found a multitude of parasite-specific miRNAs, including Onchocerca-, Loa loa-, and Brugia-derived miRNAs, in biofluids from a wide range of infected hosts, including humans (Tritten et al., 2014; Quintana et al., 2015). The potential species-specificity, and the lack of full sequence conservation of some of these miRNAs in the genome of the vertebrate hosts, make them attractive candidates as novel biomarkers for filarial infections (Quintana et al., 2015). In this regard, studies conducted on biofluids from several filarial infections suggest that, for instance, miR-71 can be used as a biomarker for filarial infection (Quintana et al., 2015). However, it is expected that several technological approaches will be considered in order to improve not only the platforms currently available for exRNA detection, but also the way in which these technologies can be transferred in a field-friendly manner (Pritchard, Cheng and Tewari, 2012; Alhassan et al., 2015). Advancing inexpensive technologies and streamlined purification and detection protocols will certainly increase the likelihood of adopting microRNA based biomarkers in the field.

Figure 1. Proposed routes of extracellular vesicles (EV) and miRNA secretion in vivo in Onchocerciasis. Depiction of a nodule-forming species member of the Onchocerca genus (e.g. Onchocerca volvulus) residing within a nodular structure termed an onchocercoma. It is not yet clear whether or how the nodular structure imposes a physical barrier for dissemination of microRNA-loaded EVs into the bloodstream. Similarly, it is unclear whether the detection of EVs and microRNAs is exclusively associated with viable worms or can be also derived from moribund or dead worms (Taken from: Quintana, Babayan and Buck, 2016).