2-Methyl-pentanoyl-carnitine (2-MPC): a urine biomarker for patent Ascaris lumbricoides infection

Abstract

Infections with intestinal worms, such as Ascaris lumbricoides, affect hundreds of millions of people in all tropical and subtropical regions of the world. Through large-scale deworming programs, World Health Organization aims to reduce moderate-to-heavy intensity infections below 1%. Current diagnosis and monitoring of these control programs are solely based on the detection of worm eggs in stool. Here we describe how metabolome analysis was used to identify the A. lumbricoides-specific urine biomarker 2-methyl pentanoyl carnitine (2-MPC). This biomarker was found to be 85.7% accurate in determining infection and 90.5% accurate in determining a moderate-to-heavy infection. Our results also demonstrate that there is a correlation between 2-MPC levels in urine and A. lumbricoides DNA detected in stool. Furthermore, the levels of 2-MPC in urine were shown to rapidly and strongly decrease upon administration of a standard treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that, although 2-MPC levels were much lower compared to humans, there was a significant association between urinary 2-MPC levels and both worm counts (p = 0.023) and the number of eggs per gram (epg) counts (p < 0.001). This report demonstrates that urinary 2-MPC can be considered an A. lumbricoides-specific biomarker that can be used to monitor infection intensity.

Figure 1

Discovery of an Ascaris lumbricoides specific biomarker. Box-and-whisker plots of the identified feature in plasma (a) and urine (b) of A. lumbricoides-positive (green, n = 38) subjects, endemic controls (red, n = 118) and healthy Belgian controls (blue, n = 40). AU = arbitrary units (intensity of ion current measured by the spectrometer).

Figure 2

Structure confirmation of 2-methyl-pentanoyl-carnitine. (a) Extracted ion chromatograms of co-injection experiment of the synthetic 2-MPC spiked to a positive plasma extract (blue) in comparison to the positive plasma extract (black). (b) MS/MS fragmentation comparison of the synthetic and natural molecules (collision-induced dissociation at 20 V). (c) IR spectra generated with infrared ion spectroscopy (IRIS) analysis on the protonated ion at m/z 260 from the HPLC purified marker (black), 2-MPC (red) and 3-MPC (blue). (d) Structure of 2-MPC.

Figure 3

Determination of a low and high cut-off for detection of infection. (a) Quantification of urinary 2-MPC in A. lumbricoides-positive (red, n = 195) subjects, endemic controls (green, n = 858) and healthy Belgian controls (blue, n = 214). Grey area indicates samples that were below lower limit of quantification (1 ng/mL). A low cut-off to identify A. lumbricoides-infected subjects was determined using ROC analysis of A. lumbricoides-positive vs. healthy controls and found to be 21.7 ng/mL. (b) Correlation between urinary 2-MPC and A. lumbricoides DNA detection in stool collected in Kenya (expressed in A. lumbricoides copies/reaction) was used to determine a high cut-off of 57.9 ng/mL 2-MPC that could be used to identify subjects with moderate-to-high infection. Moderate infection was defined as > 700 cps/rxn (see “Materials and methods” section).

Figure 4

Violin plots of the quantitative values obtained in the different populations, binned according to infection intensity (either qPCR or Kato-Katz).

Figure 5

Effect of treatment with albendazole on the presence of Ascaris lumbricoides DNA in stool (a) and urinary 2-MPC (b). Stool and urine samples were collected before (Day 0) and at different timepoints after treatment with albendazole: 6 days, 12 days and 24 days after treatment.

Figure 6

Quantification of urinary 2-MPC in A. suum infected pigs: control group, low and high trickle infected pigs (a) and correlation between urinary 2-MPC and macroscopic worms count (b) and epg count (c).

Figure 7

Presumable metabolic pathway of 2-MPC. The parasitic anaerobic carbon metabolism pathway leading to 2-methylpentanoate has been described in detail before.