Exploratory urinary metabolomics of type 1 leprosy reactions

Background: Leprosy is an infectious disease caused by Mycobacterium leprae that affects the skin and nerves. Although curable with multidrug therapy, leprosy is complicated by acute inflammatory episodes called reactions, which are the major causes of irreversible neuropathy in leprosy that occur before, during, and even after treatment. Early diagnosis and prompt treatment of reactions reduces the risk of permanent disability. Methods: This exploratory study investigated whether urinary metabolic profiles could be identified that correlate with early signs of reversal reactions (RR). A prospective cohort of leprosy patients with and without reactions and endemic controls was recruited in Nepal. Urine-derived metabolic profiles were measured longitudinally. Thus, a conventional area of biomarker identification for leprosy was extended to non-invasive urine testing. Results: It was found that the urinary metabolome could be used to discriminate endemic controls from untreated patients with mycobacterial disease. Moreover, metabolic signatures in the urine of patients developing RR were clearly different before RR onset compared to those at RR diagnosis. Conclusions: This study indicates that urinary metabolic profiles are promising host biomarkers for the detection of intra-individual changes during acute inflammation in leprosy and could contribute to early treatment and prevention of tissue damage

<i>CCL17</i> and <i>CCL18</i> by Ridley Jopling classification.

<p>Relative tissue mRNA levels of <i>CCL17</i> (A) and <i>CCL18</i> (B) normalized to GAPDH were arranged in order of specific leprosy classification and analyzed for significance by non-parametric trend test with variables TT, BT, BB, BL, and LL treated as increasing categorical variables, p values displayed. Lines are medians. A value of 40 was given to undetectable Ct Values in order to graphically represent on log scale.</p

Cytokine expression normalized by GAPDH in dermal leprosy lesions.

<p>TT+BT is tuberculoid polar leprosy, LL+BL is lepromatous polar leprosy. Values with Ct Values greater than 40 were given a value of zero and included in the statistical analysis to determine medians and 75^th percentile values. P values were calculated after Mann-Whitney analysis. Adjusted p values are the original p values corrected for multiple comparisons by traditional Bonferroni correction.</p><p>In bold are cytokines that were significant following Bonferroni correction.</p><p>*Represents cytokines that were significant by Mann-Whitney, but failed multiple comparisons corrections.</p><p>Cytokine expression normalized by GAPDH in dermal leprosy lesions.</p

Leprosy biopsy demographics.

1<p>Reversal Reaction,</p>2<p>Erythema nodosum leprosum.</p><p>Leprosy biopsy demographics.</p

CCL17 and CCL18 mRNA are not associated with reactive state.

<p>CCL17 and CCL18 mRNA levels (normalized by GAPDH expression) were compared in LL+BL versus TT+BT patients with T1R (L pole (n = 16), T pole (n = 20)) versus those without T1R (L pole (n = 17), T pole (n = 22)). Lines are medians. A value of 40 was given to undetectable Ct Values in order to graphically represent on log scale. P values were **<0.01, *<0.05, based on Mann-Whitney analysis.</p

CCL17 and CCL18 serum levels.

<p>Serum levels of CCL17 and CCL18 protein were measured in leprosy patients with lesions defined by RJ classification (n = 20) compared to EC (n = 6). P values by non-parametric trend test are depicted. EC (patients without leprosy) were given a categorical value of 0, while those with leprosy were given increasing categorical values as progressing to LL pole. Lines depict median values.</p

Cell marker expression in dermal leprosy lesions.

1<p>Tuberculoid polar leprosy,</p>2<p>lepromatous polar leprosy. Values of 0.000 are Ct values less than 1/2⁴⁰.</p>3<p>P values were calculated by Mann-Whitney tests. Both median and 75^th percentile (75(%)) are depicted.</p>4<p>Adjusted p values are the original p values corrected for multiple comparisons by traditional Bonferroni correction.</p><p>In bold are cytokines that were significant following Bonferroni correction.</p><p>*represents cytokines that were significant by Mann-Whitney, but failed multiple comparisons corrections.</p><p>Cell marker expression in dermal leprosy lesions.</p

Correlation of dermal mRNA in cutaneous leprosy lesions.

<p>Values from correlation tests (Spearman's <i>rho</i> statistics) are plotted from GAPDH normalized mRNA expression levels. Groups of probes were associated by hierarchical clustering using the complete-linkage clustering method (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003263#s2" target="_blank">methods</a>).</p