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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 21  |  Issue : 5  |  Page : 872-875
Surrogate markers of subtle renal injury in patients with visceral leishmaniasis

The Leishmaniasis Research Group/Sudan, The Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan

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Date of Web Publication31-Aug-2010


Sudanese Visceral Leishmaniasis (VL) is a disease of children that is characterized by fever, hepatosplenomegaly, lymphadenopathy, pancytopenia, and renal injury. Microalbumi要uria (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 para貞itologically 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%, res計ectively. 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;21:872-5

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 2021 Dec 4];21:872-5. Available from: https://www.sjkdt.org/text.asp?2010/21/5/872/68883

   Introduction Top

The Leishmaniases are a wide spectrum of parasitic diseases caused by flagellate protozoa belonging to the genus Leishmania of the fa衫ily Trypansomatidae. The disease pheno負es include: visceral leishmaniasisVL post kala-azar dermal leishmaniasis (PKDL), cuta要eous leishmaniasis (CL), and mucosal leish衫aniasis (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, pancyto計enia, hepato-splenomegaly, and lymphadeno計athy and can be complicated by acute renal damage. [1],[2],[3] The glomerular damage may mani苯est as mesangio-proliferative or membrano計roliferative glomerulonephritis due to depositions of immune complexes in the glomeruli. Moreover, tubulo-interstitial nephritis may occur and consists of tubular degeneration and infla衫matory infiltration with renal impairment. [1],[4],[5],[6],[7] Microalbuminuria is an early marker of glome訃ulonephritis and is defined as a persistent ele赳ation 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. [8],[9],[10]

The aim of our study is to determine the pre赳alence of renal injury in a cohort of patients with VL and evaluate it according to the pre貞ence of microalbuminuria and urRBP.

   Materials and Methods Top

Patients and samples

Following informed consent, pre-treatment serum and 24-hour urine samples were collec負ed from 88 sequential and parasitologically confirmed VL, who reported to Kassab Rural Hospital, Gedarif state, Eastern Sudan. Urine analysis, hematological profile, and serum crea負inine 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 ana衍ysis. A commercial turbidometric kit (RAN胖OX, Crumlin, Co, Antrim, UK) was used for the measurement of MA. The company's ins負ructions were strictly adhered to. One mL of reagent buffer (Polyethylene Glycol, Tris/HCL buffer, Sodium chloride) was pipetted into se赳en different test tubes (6 test tubes for stan苓ard, 1 test tube for Sample), and 0.1 mL of standards (human serum albumin) with diffe訃ent 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 spectro計hotometrically against water at 340 nm. Anti虐uman 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 stan苓ard for MA measurement) using a commer苞ial kit (ORGENTEC Diagnostika GmbH, Mainz, Germany) was used. Following removal of micro-plate strips from the pre-coated plate, calibrators, controls and undiluted urine sam計les were added to each well. Enzyme con虻ugate 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 solu負ion was added to each well, incubated for 15 minutes at room temperature. The stop solu負ion was added and the optical density was read by micro-plate reader set at 450 nm (cor訃ected by 570 nm).

urRBP was measured using sandwich enzyme衍inked immunosorbent assay (ELISA) comer苞ial kit (Immundiagnostik AG, Bensheim, Ger衫any) as follows: the pre-coated plate was washed five times with wash buffer solution. Calibrators, controls and undiluted urine sam計les 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 so衍ution. Afterwards, the diluted conjugate solu負ion was added and the plates were shaken on a horizontal mixer while incubated for one hour at room temperature. Then, the plate con負ent was decanted and the microwells were washed five times with wash buffer solution. After that, tetra-methyl benzoate substrate so衍ution was added to the wells and incubated for 10-20 minutes at room temperature. Then, the stop solution was added to each well and tho訃oughly mixed. Finally, the optical density was read at 450 nm (corrected by 570 nm).

The sensitivity, specificity, and predictive va衍ues of the immunoturbidimetry compared to ELISA for microalbuminuria measurement were calculated.

   Results Top

All of the study patients revealed within nor衫al 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, specifi苞ity, positive and negative predictive values for the turbidometric technique were calculated as 100%; 96%; 95% and 100%, respectively.

   Discussion Top

Renal damage in VL is a known complication that aggravates morbidity and increases mor負ality. 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 in苓icated by the presence of micro-albuminuria with no urinary urRBP. The presence of this renal abnormality could be attributed to infil負ration by infected macrophages and/or depo貞ition 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 crea負inine 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 immu要oturbidimetric technique is sensitive and highly specific for the detection of microalbuminuria with a markedly high negative predictive value (100%).

Pentavalent antimonials (meglumine antimo要iate 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. [1],[11],[12],[13] Recently, paromomycin (Aminosidine), which is an aminoglycoside antibiotic is under assess衫ent for the treatment of VL. [14],[15] These com binations are potentially nephrotoxic, raising concern about existing VL-induced renal da衫age and need for close follow- up. [16],[17],[18]

In conclusion, glomerular involvement is the main renal damage in patients with VL. Immu要oturbidimetric technique is a simple, sensitive and specific test with high predictive values for the presence of microalbuminuria.

   Acknowledgement Top

We wish to thank the leishmaniasis Research Group/Sudan team at the Kassab Rural Hos計ital, 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.

   References Top

1.El-Hassan AM, Khalil EA. Post-kala-azar der衫al leishmaniasis: does it play a role in the transmission of Leishmania donovani in the Sudan? Trop Med Int Health 2001;6:743-4.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]  
2.Zijlstra EE, El-Hassan AM. Leishmaniasis in Sudan. Visceral leishmaniasis. Trans R Soc Trop Med Hyg 2001;95:S27-58.  Back to cited text no. 2  [PUBMED]    
3.Khalil EA, Zijlstra EE, Kager PA, El Hassan, AM. Epidemiology and clinical manifestations of Leishmania donovani infection in two villages in an endemic area in eastern Sudan. Trop Med Int Health 2002;7:35-44.  Back to cited text no. 3      
4.van Velthuysen ML, Florquin S. Glomerulo計athy associated with parasitic infections. Clin Microbiol Rev 2000;13:55-66.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]  
5.Salgado FN, Ferreira TM, Costa JM. In赳olvement of the renal function in patients with visceral leishmaniasis (kala-azar). Rev Soc Bras Med Trop 2003;36:217-21.  Back to cited text no. 5      
6.Chaigne V, Knefati Y, Lafarge R, et al. A patient with visceral leishmaniasis and acute renal failure in necrotizing glomerulonephritis. Nephrologie 2004;25:179-83.  Back to cited text no. 6  [PUBMED]    
7.Efstratiadis G, Boura E, Giamalis P, et al. Renal involvement in a patient with visceral leishmaniasis. Nephrol Dial Transplant 2006; 21:235-6.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]  
8.Marin~ M, Andrews D, Brown D, McCluskey RT. Transcytosis of retinol-binding protein across renal proximal tubule cells after megalin (gp 330)-mediated endocytosis. J Am Soc Nephrol 2001;12:637-48.  Back to cited text no. 8      
9.Salem MA, el-Habashy SA, Saeid OM, el茅awil MM, Tawfik PH. Urinary excretion of nacetyl- beta-D-glucosaminidase and retinol binding protein as alternative indicators of nephropathy in patients with type 1 diabetes mellitus. Pediatr Diabetes 2002;3:37-41.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]  
10.Ball ST, Lapsley M, Norden AG, Cairns TD, Palmer AB, Taube DH. Urinary retinol binding protein in Indo-Asian patients with idiopathic interstitial nephritis. QJM 2003;96:363-7.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]  
11.Khalil EA, El-Hassan AM, Zijlistra EE, et al. Treatment of visceral leishmaniasis with so苓ium stibogluconate in Sudan: management ofthose who do not respond. Ann Trop Med Parasitol 1998;92:151-8.  Back to cited text no. 11      
12.Minodier P, Robert S, Retornaz K, Garnier JM. Visceral leishmaniasis: new drugs. Arch Pediatr 2003;5:550s-6s.  Back to cited text no. 12      
13.Zijlstra EE, Musa AM, Khalil EA, El-Hassan IM, El-Hassan AM. Post-kala-azar dermal leishmaniasis. Lancet Infect Dis 2003;3:87-98.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
14.Alexandridis G, Liberopoulos E, Elisaf M. Aminoglycoside-induced reversible tubular dysfunction. Pharmacology 2003;67:118-20.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]  
15.Sundar S, Jha TK, Thakur CP, Sinha PK, Bhattacharya SK. Injectable Paromomycin for Visceral Leishmaniasis in India. N Engl J Med 2007;356:2571-81.  Back to cited text no. 15      
16.Rosenthal E, Marty P. Recent understanding in the treatment of visceral leishmaniasis. J Postgrad Med 2003;49:6 1-8.  Back to cited text no. 16      
17.Cruz I, Nieto J, Moreno J, Caaavate C, Desjeux P, Alvar J. Leishmania/HIV co-infections in the second decade. Indian J Med Res 2006; 123:357-88.  Back to cited text no. 17      
18.Sundar S, Chakravarty J. Paromomycin in the treatment of leishmaniasis. Expert Opin Investig Drugs 2008;17:787-94.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]  

Correspondence Address:
EAG Khalil
Department of Clinical Pathology and Immunology, Institute of Endemic Diseases, University of Khartoum, Khartoum
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PMID: 20814123

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