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.

Urinary monocyte chemotactic protein-1 (MCP-1) in leprosy patients: increased risk for kidney damage.

BACKGROUND: We aimed to evaluate urinary MCP-1 and oxidative stress through urinary malondialdehyde (MDA) in leprosy and correlate them with traditional, but less sensitive markers of renal disease. METHODS: This is a cross-sectional study of 44 patients with diagnosis of leprosy and no previous treatment. Skin smear was assessed through a bacteriological index - from 0 to 6+. Glomerular filtration rate (GFR), protein excretion rate, microalbuminuria, urinary oxidative stress, malondialdehyde (MDA) and urinary MCP-1 were measured. Also, high- sensitivity C-reactive protein (hs-CRP) was measured in the blood. Fifteen healthy subjects composed a control group. RESULTS: Age and gender were similar between leprosy patients and control groups. No patient had a GFR < 60 mL/min/1.73 m2 or albumin excretion rate greater than 30 mg/g-Cr. Leprosy patients had higher urinary protein excretion (97.6 ± 69.2 vs. 6.5 ± 4.3 mg/g-Cr, p < 0.001), urinary MCP-1 (101.0 ± 79.8 vs. 34.5 ± 14.9 mg/g-Cr, p = 0.006) and urinary MDA levels (1.77 ± 1.31 vs. 1.27 ± 0.66 mmol/g-Cr, p = 0.0372) than healthy controls. There was a positive correlation between urinary MCP-1 and bacteriological index in skin smears (r = 0.322, p = 0.035), urinary protein excretion (r = 0.547, p < 0.001), albumin excretion rate (r = 0.414, p = 0.006) and urinary MDA (r = 0.453, p = 0.002). After adjusting for hs-CRP, urinary MCP-1 remained correlated with albumin excretion rate (rpartial = 0.483, p = 0.007) and MDA levels (rpartial = 0.555, p = 0.001). CONCLUSION: Leprosy patients with no clinical kidney disease have increased urinary MCP-1 mainly in lepromatous polar form. Inflammatory (MCP-1) and oxidative stress markers suggest leprosy patients are at high risk of developing kidney disease.

Cortisol and proinflammatory cytokine profiles in type 1 (reversal) reactions of leprosy.

PURPOSE: Cortisol levels in the circulation and at the sites of peripheral inflammation regulate type 1 (Reversal) reactions in leprosy akin to delayed type hypersensitivity reactions (DTH). In this study we determine the extent to which the differential mRNA expression of genes encoding cortisone-cortisol shuttle enzymes (11 ß hydroxysteriod dehydrogenase I & II (11 ß HSD I & II)), circulatory levels of proinflammatory cytokines (IL-6, IL-7, IP-10, IL-17F, IL-23, TNF-α, IL-1ß, PDGF BB and CRP) and cortisol are associated with development of type 1 reactions in leprosy. METHODS: Urine, blood and incisional skin biopsy samples from site of lesions were collected from 49 newly diagnosed untreated leprosy cases in T1R and 51 cases not in reaction (NR). mRNA expression levels of genes encoding 11 ß HSD I & II in skin biopsy samples were determined by realtime PCR. Cortisol levels from the lesional skin biopsies, serum and urine samples and serum proinflammatory cytokine levels were measured using ELISA. RESULTS: The mean expression ratios of 11 ß HSD I & II are significantly lower in leprosy cases with T1R when compared to the NR leprosy cases. Cortisol levels in lesional skin biopsies and in urine are significantly lower (p=0.001) in leprosy cases with T1R. Serum cytokine levels of IP-10, IL-17F, IL-IL-6 and TNF-α are significantly higher (p<0.05) in leprosy cases with T1R when compared the NR leprosy cases. CONCLUSION: Our study indicated an association of urinary and lesional skin cortisol levels with the manifestation of T1R in leprosy. IP-10, IL-17F, IL-6 and TNF-α can be potential prognostic serological markers and gene expression markers for early detection of type 1 reactions in leprosy.

High dose prednisolone treatment of leprosy patients undergoing reactions is associated with a rapid decrease in urinary nitric oxide metabolites and clinical improvement.

Evidence is accumulating that nitric oxide (NO) produced by macrophages has a role in the pathogenesis of reactions in leprosy. We followed the urinary levels of the metabolites of NO [nitrite (NO2-) and nitrate (NO3-)] and the clinical response to prednisolone treatment in leprosy patients (n = 9) admitted to ALERT leprosy hospital Addis Ababa, Ethiopia, because of reversal reaction (RR) or erythema nodosum leprosum (ENL). In untreated reactional leprosy patients, the levels of urinary NO metabolites (1645 +/- 454 microM, n = 9, ENL = 4, RR = 5) decreased significantly 2 weeks after high dose prednisolone treatment (1075 +/- 414 microM, P < 0.05), and remained stable 4 (895 +/- 385 microM, P < 0.02) and 6 weeks following treatment initiation (1048 +/- 452 microM, P < 0.02). This decrease was also present when the reactional patients were subdivided according to the type of reaction (ENL, RR) and coincided with a clinical improvement. In patients showing a poor clinical response to steroids, no or minor effects on the urinary NO metabolite levels were observed. We conclude that there is a correlation between the decrease in urinary NO metabolites and a favourable clinical response after high dose prednisolone treatment of reactional leprosy patients.

Increased levels of nitric oxide metabolites in urine from leprosy patients in reversal reaction.

We measured the metabolites of NO [nitrite (NO2-) and nitrate (NO3-)] in urine from Ethiopian patients suffering from leprosy. The urinary level of NO2-/NO3- in a group of healthy Ethiopians was 1020 +/- 471 microM (n = 22). Leprosy patients in reversal reaction had significantly higher levels of NO2-/NO3- (1817 +/- 492 microM, p < 0.001, n = 12) than both the control group and leprosy patients who were not in reversal reaction (1079 +/- 446 microM, n = 12). We conclude that the reversal reaction in leprosy in associated with increased urinary levels of nitric oxide metabolites.

Urinary excretion of renal brush border membrane enzymes in leprosy patients--effect of multidrug therapy.

Renal function at the brush border membrane level has been studied using characteristic enzymes, such as alkaline phosphatase, leucine-aminopeptidase and gamma-glutamyl transpeptidase. Urinary enzyme studies were performed using leprosy patients, classified on the basis of bacteriological index (BI>3; n=20, BI<3; n=12, BI-ve; n=10) and compared with control subjects (n=10). The role of enzymuria in monitoring WHO-recommended multidrug therapy (MDT) has been evaluated in these patients. A significant increase in the enzyme activities (p<0.01), as well as significant (p<0.01) proteinurea in 24-hour urine samples of both the smear positive groups (BI>3, BI<3) prior to therapy compared to control subjects, indicates proximal tubular functional impairment at brush border membrane level. In the smear negative (BI-ve) group, no significant difference was observed in enzyme activities as compared with the control group. In a follow-up study (BI>3;n=13, BI<3; n=4) the activities of all the enzymes decreased significantly in all the groups when compared to a corresponding untreated group. The follow-up study was not carried out on the smear negative group. The surprising finding was the differential behaviour of r-glutamyl transpeptidase, whose activity increased significantly (p<0.01) even after therapy in BI>3 group when compared with untreated patients. However in a detailed work-up including hepatic and renal function tests, the serum biochemistry was found to be normal both before and after therapy. Urinary excretion of brush border enzymes seems to be related to bacterial load, and their potential in studying the effect of MDT remains unclear.

Improving patient compliance--a multicentre evaluation of the 'DDS tile test'.

The feasibility and utility of the "DDS tile test" under field conditions was assessed in 112 leprosy centres in Maharashtra. About 10% of the 2952 urine samples tested negative for dapsone. Feed back information from 54 centres one year later showed that the test could be performed easily under field conditions and also that counselling of patients showing poor compliance helped to improve drug compliance in over 80% of cases.

Renal abnormalities in leprosy.

We have evaluated laboratory and clinical manifestations of renal disease in 96 patients with leprosy, looking for a sensitive and early marker for detection and possibly follow-up of nephropathy in these patients. Microscopic hematuria was observed in 21.9% of the cases (with dysmorphic erythrocytes in 71.4% of them). Abnormal microalbuminuria and urinary beta 2-microglobulin were found in 15.8 and 19.8% of the cases, respectively. We have observed a high frequency of hematuria, abnormal microalbuminuria and elevation of urinary beta 2-microglobulin in these patients still with normal serum creatinine.

Detection of a Mycobacterium leprae cell wall antigen in the urine of untreated and treated patients.

A total of 90 leprosy patients, 12 household contacts and 10 normal subjects were studied for the detection of Mycobacterium leprae cell wall antigen in urine using monoclonal antibody (ML30A2 IgG). In untreated multibacillary leprosy (BL-LL) the M. leprae cell wall antigen could be demonstrated in the urine of 14 (64%) patients by immunofluorescence (IF) and 22 (100%) by ELISA. In untreated paucibacillary leprosy (TT-BT), it could be demonstrated in 3 (11.5%) and in 13 (50%) patients by IF and ELISA methods respectively. All but 1 household contact (later confirmed to have BL leprosy) and all 10 normal subjects' urine was negative for M. leprae cell wall antigen by both methods. The same antigen was, however, demonstrated in urine of 50% paucibacillary patients who had received 6 months of treatment and in 68% multibacillary patients who had received 24 months of WHO recommended multidrug therapy. M. leprae cell wall antigen assays in urine will not be useful in the follow-up of leprosy patients on multidrug therapy.

[Leprosy serology: current status and perspectives]. / Sérologie de la lèpre: état actuel des connaissances et avenir.

The different serological tests used for leprosy are firstly the methods for the detection of antibodies (anti-PGL1, 35kD, 36kD, LAM), and secondly, the tests to detect the PGL1 antigen from the serum or urine. The antibody detection tests have a good but insufficient specificity for the diagnosis of leprosy patients and their sensitivity is generally high for the multibacillary patients but low for the paucibacillary patients. Their positive predictive value for the diagnosis of patients in a population are very low: 2.1% for the anti-PGL1 ELISA when the prevalence is 1/1000. For the early diagnosis of patients and the follow up of high risk populations, these tests are not cost effective: the number of patients detected in these populations is 10 fold lower than in the general population and the relative risks for developing the disease are not different among seropositive and among seronegative groups. In treated multibacillary patients, the IgM anti-PGL1 level decreases in correlation with the decrease of the bacillary index. For the diagnosis of M. leprae infection in a population, there was no correlation between the anti-PGL1 seroprevalence and the prevalence of the disease. Concerning the PGL1 antigen detection tests, they are specific and sensitive for the diagnosis of multibacillary patients but they cannot be used in routine for technical reasons. In conclusion and to date, the usefulness of serological tests in a leprosy control programme is quite questionable.

Detection of M. leprae-specific antigens with dot-ELISA in urine and nasal samples from leprosy patients.

One-hundred-two urine and nasal samples collected from leprosy patients of different classifications of disease were studied for the presence of Mycobacterium leprae antigens, including phenolic glycolipid-I (PGL-I). Lipids were extracted from the urine samples, and nasal washings were concentrated and used as such in the dot-ELISA. Two types of primary antibodies, a polyclonal antibody obtained from lepromatous (LL) leprosy patients' pooled and absorbed sera and an anti-PGL-I monoclonal antibody, were used for the detection of M. leprae antigens from these samples. The polyclonal sera detected 23% to 36% of the paucibacillary (PB) and 100% of the multibacillary (MB) leprosy cases from the urine samples. Corresponding values for nasal detection were 10% to 18% for PB and 100% for MB cases. The monoclonal antibody against PGL-I could not detect tuberculoid (TT) leprosy cases. From the urine samples, however, 16% of the borderline tuberculoid (BT), 25% of the borderline (BB), 80% of the borderline lepromatous (BL), and 100% of the LL leprosy cases were detectable. It was interesting to note that PB, skin-smear negative cases were detectable from urine examination. The specificity and sensitivity of the test is discussed in relation to the crossreacting antigens.

Comparison of a urine spot test and blood tests as indicators of patient compliance.

Irregular drug intake has been a concern of leprosy control programmes for many years and various methods have been used to monitor and encourage patient compliance. This study compares the results of a urine spot test for dapsone as proposed by Huikeshoven, with blood levels measured in the same patients by the modified Bratton Marshall method and by high performance liquid chromatography. Two hundred-sixty urine and blood specimens were obtained from subjects who were taking supervised and unsupervised medications as well as from controls who were taking no medications. The results indicate that the urine spot test is simple and easily performed, and for monitoring patient compliance under routine clinical conditions (hospital or field work) it compares favourably with blood levels of dapsone estimated by the Bratton Marshall method or by high performance liquid chromatography. The study also shows that dapsone level is not a good indicator of compliance in patients who are also taking daily rifampicin but the urine spot test remains useful in such patients.

A method for the detection of M. leprae antigens in urine of leprosy patients by gel diffusion and gel electrophoretic techniques and significance in diagnosis.

A simple and novel method has been developed for the first time to detect M. leprae antigens in the urine of leprosy patients by treating the urine samples with sodium dodecyl sulphate (SDS). The antigens thus released can be demonstrated by simpler techniques like gel diffusion. By this method about 86% antigen positivity is observed in the urine of TT/BT and 83% positivity in BB patients. In BL/LL patients the antigen positivity is observed in 87% of the subjects. The high rate of M. leprae antigen positivity by the present method in the urine of patients with early leprosy may prove to be of diagnostic significance.

Interpreting complicated chromatographic patterns.

GC-MS, HPLC, automatic amino acid analysis, high-resolution two-dimensional electrophoresis and capillary electrophoresis are suitable for the multicomponent analysis of body fluids and tissues. Manual interpretation of the complex metabolite and protein profiles thereby obtained is usually difficult, except in the case of metabolic disorders, where major deviations from the normal profiles often are observed. Implementation of multivariate data analysis makes it possible to retrieve diagnostic information that otherwise may be overlooked, as shown in this report where patients with leprosy have been examined. Urine samples were analysed by ion-exchange chromatography and by GC-MS to obtain profiles of amino acids and organic acids. Qualitative and quantitative information on 68 metabolites were then analysed by principal components analysis (PCA) and by partial least square models (PLS). Three different PLS dimensions were found (cross-validation) corresponding to controls (persons without leprosy), paucibacillary and multibacillary leprosy.

Pattern of drug compliance in leprosy patients attending urban centres--a longitudinal study.

The pattern of drug compliance in 485 leprosy patients attending urban leprosy centres in Bombay was studied for 2 years. The study subjects included 113 patients with paucibacillary leprosy under dapsone monotherapy, 241 patients with paucibacillary leprosy under multidrug therapy and 131 patients with multibacillary leprosy under multidrug therapy. Their urine samples had been checked at least 6 times during the 2 years by DDS tile test at the time of their clinic attendance. The urine test results were not disclosed to the patients, but patients showing negative results were counselled about the need for regular drug intake. 35% of the patients were "Regular through out", 13% were "Irregular through out" and the other 52% who "Tended to be irregular" in their drug intake became "Regular" after counselling. Regularity in drug compliance was better in patients on multidrug therapy than in those on monotherapy. It is suggested that periodic testing of urine for checking for regularity of drug intake and subsequent counselling of patients should be made a routine practice to maintain drug compliance at a high level.

Dapsone drug compliance study among leprosy patients: a comparison between qualitative and quantitative methods.

The methods currently employed to monitor self-administration of dapsone have been evaluated by comparing the results of the qualitative spot test and quantitative DDS/creatinine ratio test. Random urine samples of 242 leprosy patients, periodically attending the Leprosy Clinic were tested. Although a good correlation between the results of the two tests was evident, the DDS/creatinine ratio technique appeared to be more sensitive than the spot test. The concentration of DDS and its metabolites in urine specimens found to be negative by the spot test, ranged from 3.32-12.37 micrograms of DDS/mg creatinine. The spot test was found to be more specific and stays to be the method of choice, when rapidity and reproducibility are the prime objectives, and sensitivity can be marginally compromised. Acidification of urine prior to the spot test was found to be desirable to rule out false negative and false positive reactions.