Parasite-derived microRNAs in host serum as novel biomarkers of helminth infection

Abstract

Background: MicroRNAs (miRNAs) are a class of short non-coding RNA that play important roles in disease processes in animals and are present in a highly stable cell-free form in body fluids. Here, we examine the capacity of host and parasite miRNAs to serve as tissue or serum biomarkers of Schistosoma mansoni infection.

Methods/principal findings: We used Exiqon miRNA microarrays to profile miRNA expression in the livers of mice infected with S. mansoni at 7 weeks post-infection. Thirty-three mouse miRNAs were differentially expressed in infected compared to naïve mice (>2 fold change, p<0.05) including miR-199a-3p, miR-199a-5p, miR-214 and miR-21, which have previously been associated with liver fibrosis in other settings. Five of the mouse miRNAs were also significantly elevated in serum by twelve weeks post-infection. Sequencing of small RNAs from serum confirmed the presence of these miRNAs and further revealed eleven parasite-derived miRNAs that were detectable by eight weeks post infection. Analysis of host and parasite miRNA abundance by qRT-PCR was extended to serum of patients from low and high infection sites in Zimbabwe and Uganda. The host-derived miRNAs failed to distinguish uninfected from infected individuals. However, analysis of three of the parasite-derived miRNAs (miR-277, miR-3479-3p and bantam) could detect infected individuals from low and high infection intensity sites with specificity/sensitivity values of 89%/80% and 80%/90%, respectively.

Conclusions: This work identifies parasite-derived miRNAs as novel markers of S. mansoni infection in both mice and humans, with the potential to be used with existing techniques to improve S. mansoni diagnosis. In contrast, although host miRNAs are differentially expressed in the liver during infection their abundance levels in serum are variable in human patients and may be useful in cases of extreme pathology but likely hold limited value for detecting prevalence of infection.

Figure 1. Differential expression of host miRNAs in the livers of mice at 4–12 weeks post infection with S. mansoni.

miRNAs were quantified by qRT-PCR, normalized to miR-16 and fold changes calculated as the ratio of values from infected versus naïve mice (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 2. Differential abundance of host miRNAs in mouse serum during S. mansoni infection.

miRNAs were quantified by qRT-PCR and normalized to a synthetic RNA spike-in. Each symbol represents data from one individual mouse. Fold changes are defined as the ratio of abundance in infected versus naïve serum; the signal from naïve was set as 1. (***p<0.001, ****p<0.0001).

Figure 3. Predicted stem-loop structures of the S. mansoni miRNAs identified in this study.

The mature sequence that was identified in serum is shown in bold.

Figure 4. Detection of parasite-derived miRNAs in mouse serum during S. mansoni infection.

miRNAs were quantified by qRT-PCR, normalized to a synthetic RNA spike-in and fold change calculated as the ratio of abundance in infected serum compared to the background abundance level detected in naïve serum, which represents the noise in the assay likely derived from cross-hybridization with endogenous small RNAs (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 5. Discrimination between the S. mansoni infected and uninfected individuals using parasite-specific miRNA detection.

miRNAs were quantified by qRT-PCR, normalized to a synthetic RNA spike-in and fold changes calculated as the ratio of infected to uninfected (median with interquartile range indicated). Piida- panel A, Chiredzi- panel B. Specificity and sensitivity and ROC curves with AUC are indicated. (*p<0.05, **p<0.01).

Figure 6. Discrimination between the S. mansoni infected and uninfected individuals using the combined data for 3 parasite miRNAs.

Fold-changes (median with interquartile range indicated) and ROC curves for bantam, miR-277 and miR-3479-3p (Piida- panel A, Chiredzi-panel B) (**p<0.01).