Use of the polymerase chain reaction to detect Mycobacterium leprae in urine

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients’ urine samples was successfully amplified by PCR-Pra in 46.6 percent (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75 percent for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30 percent for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

Use of the polymerase chain reaction to detect Mycobacterium leprae in urine.

Leprosy is an infectious disease caused by Mycobacterium leprae. The polymerase chain reaction (PCR) has been applied to detect M. leprae in different clinical samples and urine seems to be attractive for this purpose. PCR was used to improve the sensitivity for diagnosing leprosy by amplifying a 151-bp PCR fragment of the M. leprae pra gene (PCR-Pra) in urine samples. Seventy-three leprosy patients (39 males and 34 females, 14 to 78 years old) were selected for leprosy diagnosis at a reference laboratory in Maringá, PR, Brazil. Of these, 36 were under anti-leprosy multidrug therapy with dapsone and rifampicin for tuberculoid (TT) and dapsone, rifampicin and clofazimine for borderline (BB) and lepromatous (LL) forms. The control group contained 50 healthy individuals without any clinical history of leprosy. DNA isolated from leprosy patients' urine samples was successfully amplified by PCR-Pra in 46.6% (34/73) of the cases. The positivity of PCR-Pra for patients with the TT form was 75% for both patients under treatment and non-treated patients (P = 0.1306). In patients with the LL form, PCR-Pra positivity was 52 and 30% for patients under treatment and non-treated patients, respectively (P = 0.2386). PCR-Pra showed a statistically significant difference in detecting M. leprae between the TT and LL forms of leprosy in patients under treatment (P = 0.0033). Although the current study showed that the proposed PCR-Pra has some limitations in the detection of M. leprae, this method has the potential to be a useful tool for leprosy diagnosis mainly in TT leprosy where the AFB slit-skin smear is always negative.

Increased level of urinary nitric oxide metabolites in leprosy patients during type 2 reactions and decreased after antireactional therapy.

OBJECTIVES: To assess the urinary nitric oxide metabolites in lepromatous patients in ENL (type 2 reactions) and to compare these metabolites after subsidence of reactions following antireactional therapy. Further to compare the levels in a group of lepromatous leprosy patients without reactions. DESIGN: The initial urine samples were collected from lepromatous leprosy patients when they came with ENL before commencing antireactional therapy and repeat samples were taken after resolution of ENL. Morning urine samples were collected from LL patients without reactions. Nitrites and nitrates in urine were measured using commercially available kit. Mean levels of nitric oxide metabolites of LL patients with ENL and without ENL were compared by student's 't' test. The level during ENL and after resolution was compared by paired 't' test. RESULTS: The nitric oxide metabolites were analyzed in 14 LL patients with ENL and after resolution of ENL and in 5 LL patients without reaction. The level of urinary nitric oxide metabolite is higher in LL patients in ENL reaction compared to LL patients without reaction (P < 0.04). These levels were reduced significantly with resolution of reaction following antireactional therapy (P < 0.004). CONCLUSION: The findings of this study suggested that the NO/NOM excretion is increased in leprosy patients during ENL episodes. With antireactional therapy (steroids) and clinical improvement the levels are reduced.

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.

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.

Urinary excretion of renal brush-border enzymes in lepromatous leprosy--a preliminary investigation.

Activities of the brush-border enzymes, alkaline phosphatase, maltase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, were measured in urine samples of 25 lepromatous leprosy patients and an equal number of age-matched healthy controls. None of the patients were shown to be suffering from any other systematic disease. The enzymatic activities were shown to be significantly elevated in leprosy patients when compared to controls.

Urinary excretion of renal brush-border enzymes in lepromatous leprosy - a preliminary investigation

Activities of the brush-border enzymes, alkaline phosphatase, maltase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, were measured in urine samples of 25 lepromatous leprosy patients and an equal number of age-matched healthy controls. None of the patients were shown to be suffering from any other systematic disease. The enzymatic activities were shown to be significantly elevated in leprosy patients when compared to controls

Detection of leprosy antigenuria through DOT-ELISA.

Fifty-five samples of urine from different grades of leprosy patients and normal persons were processed for detection of PGL-1 antigen through DOT-ELISA on nitrocellulose paper strips using anti-human IgG horse raddish peroxidaseconjugate. About 66.6% of the paucibacillary and 100% of the multibacillary leprosy cases were detectable through this technique on the basis of differential colour development on the strips. Possibility of its use in the field conditions has been discussed.