Year : 2010 | Volume
: 21 | Issue : 5 | Page : 872--875
Surrogate markers of subtle renal injury in patients with visceral leishmaniasis
NAA Elnojomi, AM Musa, BM Younis, MEE Elfaki, AM El-Hassan, EAG Khalil
The Leishmaniasis Research Group/Sudan, The Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan
Department of Clinical Pathology and Immunology, Institute of Endemic Diseases, University of Khartoum, Khartoum
Sudanese Visceral Leishmaniasis (VL) is a disease of children that is characterized by fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, and renal injury. Microalbuminuria (MA) and urinary retinol binding protein (urRBP) are useful markers for glomerular and tubular dysfunctions, respectively. We report the prevalence of subtle renal injury in 88 parasitologically confirmed VL patients in a cross-sectional and hospital-based study. Blood and urine were collected before treatment for hematological, biochemical profiles in addition to MA and urRBP measurement using competitive solid phase, sandwich enzyme-linked immune sorbent assay (ELISA), and immunoturbidometry. All the patients had normal serum urea and creatinine levels and no detectable urRBP. However, 40% of the patients had MA detected by ELISA, and 42% were reactive with turbidometry. The sensitivity, specificity, positive and negative predictive values for MA turbidometric technique were calculated as 100%; 96%; 95% and 100%, respectively. In conclusion; subtle renal injury in VL is mainly glomerular. Turbidometry for MA measurement is a simple, inexpensive, sensitive, and specific technique with high predictive values.
|How to cite this article:|
Elnojomi N, Musa A M, Younis B M, Elfaki M, El-Hassan A M, Khalil E. Surrogate markers of subtle renal injury in patients with visceral leishmaniasis.Saudi J Kidney Dis Transpl 2010;21:872-875
|How to cite this URL:|
Elnojomi N, Musa A M, Younis B M, Elfaki M, El-Hassan A M, Khalil E. Surrogate markers of subtle renal injury in patients with visceral leishmaniasis. Saudi J Kidney Dis Transpl [serial online] 2010 [cited 2020 Sep 21 ];21:872-875
Available from: http://www.sjkdt.org/text.asp?2010/21/5/872/68883
The Leishmaniases are a wide spectrum of parasitic diseases caused by flagellate protozoa belonging to the genus Leishmania of the family Trypansomatidae. The disease phenotes include: visceral leishmaniasisVL post kala-azar dermal leishmaniasis (PKDL), cutaneous leishmaniasis (CL), and mucosal leishmaniasis (ML). Visceral Leishmaniasis (VL), caused by Leishmania donovani is the most serious, mainly affects children, and is fatal if untreated. The disease is prevalent over large areas of Sudan with an incidence rate of 6080/1000-individuals/year. VL is a febrile illness that is characterized by weight loss, pancytopenia, hepato-splenomegaly, and lymphadenopathy and can be complicated by acute renal damage. ,, The glomerular damage may manifest as mesangio-proliferative or membranoproliferative glomerulonephritis due to depositions of immune complexes in the glomeruli. Moreover, tubulo-interstitial nephritis may occur and consists of tubular degeneration and inflammatory infiltration with renal impairment. ,,,, Microalbuminuria is an early marker of glomerulonephritis and is defined as a persistent elevation of albumin in urine of 30-300 mg/day. On the other hand, tubulo-interstitial nephritis and proximal tubulopathies are associated with excretion of urinary retinol binding protein (urRBP), a low molecular weight protein that is completely reabsorbed by the kidneys, > 400 μg/L. ,,
The aim of our study is to determine the prevalence of renal injury in a cohort of patients with VL and evaluate it according to the presence of microalbuminuria and urRBP.
Materials and Methods
Patients and samples
Following informed consent, pre-treatment serum and 24-hour urine samples were collected from 88 sequential and parasitologically confirmed VL, who reported to Kassab Rural Hospital, Gedarif state, Eastern Sudan. Urine analysis, hematological profile, and serum creatinine and urea were measured for all patients.
Microalbuminuria and urRBP measurement
24-hour urine was collected in clean plastic containers with boric acid as preservative. The urine samples were kept in the fridge till analysis. A commercial turbidometric kit (RANDOX, Crumlin, Co, Antrim, UK) was used for the measurement of MA. The company's instructions were strictly adhered to. One mL of reagent buffer (Polyethylene Glycol, Tris/HCL buffer, Sodium chloride) was pipetted into seven different test tubes (6 test tubes for standard, 1 test tube for Sample), and 0.1 mL of standards (human serum albumin) with different concentrations (5, 10, 20, 50, 100, 200 μg) and 0.1 mL of urine samples were added into the test tubes. The tubes were thoroughly mixed, and the initial absorbance was read spectrophotometrically against water at 340 nm. Antihuman albumin antibody (0.1 mL) was added, the tubes were thoroughly mixed and covered with plastic cling film and incubated for 30 minutes at 15-25ºC. The final absorbance of each solution was read spectrophotometrically at 340 nm, and the concentrations were calculated from the constructed standard curve.
Competitive solid phase ELISA (gold standard for MA measurement) using a commercial kit (ORGENTEC Diagnostika GmbH, Mainz, Germany) was used. Following removal of micro-plate strips from the pre-coated plate, calibrators, controls and undiluted urine samples were added to each well. Enzyme conjugate solution (polyclonal rabbit anti-human albumin labeled with horseradish peroxidase) was added to each well and incubated for 30 minutes at room temperature. The contents of the well were discarded, and the micro-wells were washed three times with wash buffer solution. Tetra-methyl benzoate substrate solution was added to each well, incubated for 15 minutes at room temperature. The stop solution was added and the optical density was read by micro-plate reader set at 450 nm (corrected by 570 nm).
urRBP was measured using sandwich enzymelinked immunosorbent assay (ELISA) comercial kit (Immundiagnostik AG, Bensheim, Germany) as follows: the pre-coated plate was washed five times with wash buffer solution. Calibrators, controls and undiluted urine samples were added to each well, incubated for one hour at room temperature. The content of the wells were decanted and the micro-wells were washed five times with wash buffer solution. Afterwards, the diluted conjugate solution was added and the plates were shaken on a horizontal mixer while incubated for one hour at room temperature. Then, the plate content was decanted and the microwells were washed five times with wash buffer solution. After that, tetra-methyl benzoate substrate solution was added to the wells and incubated for 10-20 minutes at room temperature. Then, the stop solution was added to each well and thoroughly mixed. Finally, the optical density was read at 450 nm (corrected by 570 nm).
The sensitivity, specificity, and predictive values of the immunoturbidimetry compared to ELISA for microalbuminuria measurement were calculated.
All of the study patients revealed within normal range serum urea and creatinine levels. The routine urinalysis showed no albuminuria or increased pus cells. However, 35/88 (40%) of the study VL patients had detectable MA by the ELISA technique. Turbidometrically, MA was detected in 42% (37/88) of patients. urRBP could not be detected in any of the samples using ELISA. The sensitivity, specificity, positive and negative predictive values for the turbidometric technique were calculated as 100%; 96%; 95% and 100%, respectively.
Renal damage in VL is a known complication that aggravates morbidity and increases mortality. In Sudan, VL mostly affects children in remote areas, where health facilities are meager and laboratory investigations are non-existent. Subtle renal involvement in Sudanese patients with VL is mainly glomerular in nature as indicated by the presence of micro-albuminuria with no urinary urRBP. The presence of this renal abnormality could be attributed to infiltration by infected macrophages and/or deposition of immune complexes in the glomeruli.
The results of the present study showed that more than 40% of VL patients present with subtle renal glomerular damage as indicated by the presence of significant microalbuminuria but normal serum urea and creatinine. In the present study, normal serum urea and creatinine levels probably point to the crudeness of these tests as indicators of renal damage. Since, drug-combination therapy for VL is the trend worldwide, more sensitive investigations are needed to differentiate from iatrogenic renal damage.
The present study documented that the immunoturbidimetric technique is sensitive and highly specific for the detection of microalbuminuria with a markedly high negative predictive value (100%).
Pentavalent antimonials (meglumine antimoniate and sodium stibogluconate) are the first line of treatment; they are toxic and have to be used parentrally for prolonged periods. Although second-line drugs (e.g liposomal amphotericin B) are less toxic, they are very expensive. ,,, Recently, paromomycin (Aminosidine®), which is an aminoglycoside antibiotic is under assessment for the treatment of VL. , These com binations are potentially nephrotoxic, raising concern about existing VL-induced renal damage and need for close follow- up. ,,
In conclusion, glomerular involvement is the main renal damage in patients with VL. Immunoturbidimetric technique is a simple, sensitive and specific test with high predictive values for the presence of microalbuminuria.
We wish to thank the leishmaniasis Research Group/Sudan team at the Kassab Rural Hospital, Gedarif State, Eastern Sudan. This study was supported by a grant from the Department of Clinical Pathology & Immunology, Institute of Endemic Diseases, University of Khartoum.
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