IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Evaluation o f Dot. ELISA test for Diagnosis Visceral Leishmaniasis in Infected Children W. R. Ali , R. S. Al-Sady and A. A. J. J. Al- Saadi Departme nt of Biology ,College of Education Ibn-Al-Haitham, Unive rsity of Baghdad Abstract For t he first time in Iraq, this study was conducted to evaluate the usefulness of Dot .ELISA, for detecting anti - Leishmania donovani antibodies in serum samples from susp ected p atient (children under 8 y ears ) with Visceral Leishmaniasis V.L.. Sera from 73 V.L. , 60 Healthy controls, and 57 p atient with ot her parasitic diseases ot her than V.L. (Amoebiasis, Giardiasis , Toxop lasmosis, Schist osomiasis , Hy datidosis, Ascariasis , Lupus Erythromatosus , Viral Hep atitis, and Cutaneous Leishmaniasis) were examined. Ant i Leishmania donovani antibodies detected in 71 out of 73 susp ected Visceral Leishmaniasis . Data of this st udy showed that infection in male group was more than female group . Result of this st udy revealed a high p revalence of p atients with V.L. infection at the age range 1 - 2.9 and 3 - 4.9 y ears when comp ared with other range group s (P>0.01). A cross – reaction was found with sera Lupus Erythromatosus, No cross - reaction was found with sera from other disease . Sensitivity was determined as 97.3 %, and Sp ecificity was found to be 95.7% . Positive and Negative the Values Predictive of test were 96% and 97.4% Dot .ELISA is simp ler, Serodiagnosis Rap id, sensitive and sp ecific, Its use in resp ectively. Seeing that of V.L. can be recommended. Introduction Visceral Leishmaniasis V.L. the second largest p arasite killer in the world (after malaria)which is resp onsible for an estimated 500,000 new cases of V.L. occur in each y ear and a tenth of these p atients would die [1]. In Iraq esp ecially V.L. was regarded as an endemic disease[2].Demonstration of p arasites in bone marrow asp irates or needle biopsy sp ecimens of the sp leen, and lymph node, or by vitro cultivation are the definitive methods of diagnosis [3]. However, these methods are insufficiently sensitive, and the techniques are invasive, p ainful , and even hazardous [4]. A number of serological tests have been developed and evaluated for the diagnosis of V.L., DAT [5] and ELISA [6, 7]. Later flow using recombinant antigen [7,8] and PCR [9].In Iraq, various epidemiological survey of V.L. was done by using IFAT technique [10]. This test requires more exp ensive and sp ecialized equip ments and it is not suitable for large scale examination of sera [11]. Various ty p es of ELISA has been developed and described for comp etition , the direct method for detection antibody [ 6 ] and the double antibody sandwich method for detection antigen [7]. It is usful in field owimg to its simplicity Dot .ELISA has been succefully app lied to the Serodiagnosis of p arasitic diseases, because it is simple to p erform, inexp ensive , and highly accurate and serum conservative [ 5 ]. The present st udy aimed to evaluate the Dot . ELISA for Serodiagnosis of V.L. by using Promastigote antigen and human sera infected with V.L. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Material and Methods Venous blood samp les were collected from 73 Children under Eight y ears susp ected to have V.L. from Ch ild ' s Central Teachin g Hosp ital in Baghdad during Ap ril- September 2007. V.L. was diagnosed on the basi c Le ishmania serology (IFATor Dip stck) . leishman donov an bodies were detected bone marrow asp irates . Control serum samples were obtained from 6o Health subjects with no known exp osure to V.L. . Cross reactivity were performed by using 57 samples from patient with p arasitic infection than V. L. , Including Amoebiasis (n=5) , Giardiasis (n=5) , Toxop lasmosis (n=5) ,Schist osomiasis (n=5), Hy datidosis (n=5), Ascariasis (n=5) , Viral Hep atitis (A=5/B=2) , Lup us Erythromatosus (n=5), and Cutaneous Leishmaniasis (n=15). Preparation of Antigen The p rimary isolation of p arasites from bone marrow asp irates of susp ected V.L. patients were grown in semisolid medium which p repared according Limoncou etal., [12]. Some of the asp irates were inoculated in Novey , M ac Neal, Nicol (NNN) medium at 25°C to isolate, and culture visceral st ock [9]. Aft er the Promastigotes of Leishmania donovani growing with 7-10 days [13] on Semisolid medium were transferred to 100ml st erile containers, and then 10ml of liquid medium was added [14]. The container was incubated at 25°C in orbital incubator. The growt h of p arasites were examined after two days to ensure a good growt h and sterilely. Ot her quantities of liquid medium were added when needed. The p arasites were amp lified on liquid medium after 3-5 days later, a drop of culture were examined to check the p resence of , sufficient number of Amastigote . The culture characterist ically contains ≥97% motile Promastigotes. The Promastigotes were collected by centrifuging the liquid medium overlay at 750g for 15 min at 4 °C. The pellets were washed three times with PBS, parasites fixed in 50ml of 1.5% formalin - PBS fore 1hr. at 22°C and washed three times in Triethanol amine Buffered Saline(TBS),p H7.5, and estimation of the p rotein content of antigen to the method of Bradford [15]. Dot.ELIS A procedure: Dot . ELISA was p erformed as described by Pap p as etal., [16 ]. A nitrocellulose filter discs (25 mm diameter,0.22µl p ore size, M illip or,Inc., Bedford.M A) cut into 5 x5mm square, and all discs are handled with forcep s to p revent contamination. 1 µl Promastigotes antigen (4.2 x 10 5 Promastigotes, 1 µg p rotein ) was dot ted onto each disc by10 µl Hamilton sy ringe . Antigen was st abilized on the discs by dry ing for 10min.at 56°C. Ant igen discs were then st ored at -20°C until used [16 ]. All st eps were p reformed at room temp erature 22-24 °C an all working solution were made in TBS. Before antigen discs were left in room temp erature, then p laced in wells of flat-bot tom 96-well micro liter plates (M axi Sorp ., Nune, Roskilde, Denmark). Un bound sites on the discs were blocked with 75 µl of 5% of Bovine serum albumin BSA – TBS (w/v).The micro titer p lates were shaken fore one minute, then incubated for 15 min. The p rocedure adequately blocked all nonsp ecific p rotein binding sites on antigen discs and all surfaces. Blocking solution was asp irated off the antigen discs and 50 µl of 4 fold dilution of saea in 1% BSA -TBS (w/v)was added to each well. Plates shaken for 1min. and incubated for 30min. Sera were asp irated off and washed three times in 100 µl of 0.05 Nonided P-40 ( NP- 40.PH6.85,was p urcosed from Sheel Chemical Co., Ltd. West Orange, NJ). During the third wash antigen discs were incubated in NP-40-TBS for 10 min. before asp iration 50 µl hors radish p eroxidase (HRP)conjugated goat antihuman IgG (Sigma Chemical) diluted 1:100 in 1% BSA – TBS ( p H7.4 ) was p ipett e in 50 µl volumes into each well and the p lates were shaken then incubated for 30min. conjugated asp irated off all test samples and the p lates were washed as described above. Freshly p repared substrate by mixing together 2ml of 4- Chloro – 1- nap hthol IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 4CIN, a p ricipitable substrate , was dissolved in anhydrous methanol (3 mg / ml ) and st ored in a brown bottle in the dark at 4°C . Every 14 days this st ock solution was p repared fresh. Immediately to be for use, 10ml of TBS and 4 µl of 30% H202 were added to 2 ml of 4CIN. Fifty micro liters of t his solution (PH 7.35 - 7.4 ) were added to each test well and the p lates were shaken and incubated for 30min. Subst rate was asp irated and the p lates were washed three times in TBS and were allowed to dry . Serum dilutions causing the develop ment of well- defined blue dots on antigen discs were considered positive. S tatisti cal analysi s: All the st atist ical analysis were done by zar [17]. Results Table (1) showed that 71 out of 73samples from suspected V.L. male p atients 46 , 64.8 were higher than number and p ercentage of female p atients 25, 35.2 %. Data demonst rated in Figure(2) showed that the different p revalence of V.L. in patients with represent t o age group s range 1-2.9 and 3-4.9 y ears (46.57% ,n=33) and (26.8%, n=19) were resp ectively more p revalence of si gnificance was (P>0.05). For Dot .ELI SA , 71 sera fro m susp ected p atients, blue dot were observed against 4 fold dilutions of sera. Al l the control sera (60 cases ) gave negative results. A cross – reaction was observed with serum samp les from p atients with Lupus Erythromatosus .Whereas as serum samples from p atients with (Amoebiasis , Giardiasis, Toxop lasmosis ,Schist osomiasis , Hy datidosis , Ascariasis , Viral Hep atitis , and Cutaneous Leishman iasis ) didn't cross react.Sensitive of 97.3%(71 p ositive of 73 V.L. p atient samp les ), and the sp ecificity was found to be 95.7% (112 negatives of 117 non p atients samp les ).Considerin g the above findin gs, T herefore p ositive and negative predictive valu es of the assay were 96%,97.4% resp ectively. Discussion Numerous epidemiological and clinical st udies had noted difference in the p revalence of Leishmania disease between males and females in Iraq[10], Al-jeboor i [18] found that male : females was 3:1 in the p art central p art of Iraq. There were nu merous observations whi ch demonst rated that t he difference in the prevalence of V.L. disease between males and f emales was related to the difference in the host immune resp onse to infection p lay ed an imp ortant role in the resistance and susceptibility of infection due to increased exp ression of INF -gamma in females in comp arison with males which led to increased female protection against the infection [19]. V.L. occurs mostly in y oung p op ulation. The results showed that the p revalence of the disease described in p atients with increasin g of age. Similar results were obtain ed in other st udied in Iraq and neighboring countries [10, 20, 21, and 22]. These findings might be due to difference in the host immune r esp onse ( humoral and cell mediated immune r esp onse), that will be considered infant is not well develop ed at an early st age of life. The high er rate at age of 1-2.9 y ears might be due to the difference in suscep tibility of p op ulation to the infection. Anot her p ossible cause for the infection of the infants was that the sand flies which have bett er opp ortunities of biting relatively immobile infant than other children [23]. The Dot. ELISA has been successfully app lied in Serodiagnosis of many p arasitic diseases and has several advantages [24]. The results can be read with naked eye and the antigen dotted nitrocellulose membrane st rips can be st ored for 3 y ears at 4°C. As demonst rated in Yamaura and Arak st udy [25], it has been also reported that antigen – dotted nitrocellulose membrane st ored for up to 3 months at room temp erature 37°C. The sensitivity of the Dot. ELISA in the present study 97.3 % and sp ecificity was 95.7 % in the diagnosis of V.L. p ositive negative predictive values were 96 % and 97.4 % resp ectively. Our data agreed with Dietz e study [26] which improved that Dot . ELISA t est with high IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 sensitivity of 97% and sp ecificity of 100 % in diagnosis of Canine V.L. Our finding is consist with Ali st udy [24] in Iraq , which found that sensitive 97.3% and sp ecific 95.7% for the diagnosis of human Hy datidosis. Hatam etal., [27] used Dot . ELISA to Serodiagnosis of Amoeboasis was found to be more sp ecific 96.3% and sensitivity 83.3 % , Positive and Negative Predicative values were 93.7% and 89.6% resp ectively. Cross- reactions were observed between test antigen and sera from 5 p atients with Lupus Ery thromatosus. This was most likely due to lupus p atients anti – nuclear antibodies reaction with p arasite DNA. T hese cross reactions were however, not high cross- reactivity with sera from p atients with confirmed Cutaneous Leishmaniasis was not observed .This is most likely due to the observation that serum antibody levels are characterist ically lower in cases of Cutaneous Leishmaniasis [16]. Dot . ELISA had a higher sp ecificity and sensitivity and since it is a simp le , no exp ensive and reliable test , its use can be recommended. Re ferences 1- Boelaert , M . ; Criel , B. ; Leewenburg , J. ; Dammevan , W. and Leray , D. (2000) . Trans. R. soc. Trop . M ed. Hy g., 94: 465-471. 2- AL- Nadawi , M . N. ; AL-Safar, ,N. and AL-Ezz i ,J. (2000). J. Fac. Bag. M ed.Bag.,42(2):95-99. 3-M arsden, P.D.(1979). Engl. J. M ed., 300 : 350-352. 4- Kar, K. (1995). Crit. Rev. M icobiol., 21:123-152. 5-Bagchi , A. K. ; Tiwari ,S. ; Gup ta , A. and Schoone , G. (1998). Ann. Trop . M ed. Parasitol., 92: 154-163. 6- Rajasekar iah , G. H. R. ;Cardoso , l. ; Do gcio , D. A. ; M artin , S.K. and Smithy man A. M . (2008). Am. J. T rop . M ed. Hy g., 78(4) : 616-623. 7- Hommel , M .(1976). Trans. Roy . Soc. Trop . M ed. Hy g. , 70: 15-16. 8- Qu, J. Q. ; Zhong , L. ; M asoom - Yasin Zai , M . ; Abdure – Rab , M . ; Aksu , H. S. ; Reed , S. G. ; Chang , K. P. and Gilman - Sachs , A. (1994). 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T. (1984). J. Immunol.M eth.,64:205-214. 17-Zar,J. N. (1999). Biost atist ical analysis. 4 th ed. Prentice-Hall, Inc., USA. 18- AL-Jeboori ,T. L. (1979). Trans. Roy . Soc. Trop . M ed. Hy g. 75: 109. 19- Islam ,M . Z. ; Itoh ,M . ; Shamsuz zaman , S. M . ; M irza ,R. ; M atin .F. ; Ahmed ,I. ; Choudhury , A. K. M . S. ; Hussain , A. H. ; QiU, Xu-G. ;Begam , N. ; Furuy a ,M . ; Leafasia , J. L. ; Hashiguchi ,Y. and Kimura , E.,(2002).Clin. Diagh. Lab.Immunol.,9(4):789-794. 20-Alwan , S. J. (1985) . The p resent st atus of visceral Leishmaniasis in Baghdad City . M . SC. Thesis .Coll. M ed., Univ. Baghdad. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 21- Al-ALak , S. F.(1997). Study of epidemiology of V.L. (Kala- azar ) in AL-M agger Dist rict– M issanp rovince. M . SC. Thesis, College of M edicine, University of Baghdad. 22- Satoskar , A. ; AL- Quassi , H.H. and Alexander , J. (1998).. Immunol. Cell.Biol., 76 : 159 -166. 23-Bray , R. S. (1974) . Leishmaniasis. Ann . Rev. M icrobiol., 28 : 189-210. 24–Ali,W. R. (2008). IBN AL-Haitham J. Pure & Ap p l. Sci., 21(4):1-11. 25- Yamaura , H. ; Araki , K. ; Kikuchi , K. ; Itoda , I. ; Tot suka , K .; Kobay akwa ,T. (2003). Am. Trop . M ed. Hy g., 59: 431- 434. 26-Dietz e, R. ; Falqueto, A. ; Valli, L. C. P. ; Rodriques, T. P. ; Boulos , M . and Corey, R. (1995). Ann. J. Trop . M ed. Hy g., 53 (1): 40-42. 27-Hatam ,Gh ; Khormi , HR. ; Sahebani ,N. and Sarkari ,B.(2007).ShirazE-M ed.J.,8(3):1-9. Table (1) Distribution of V.L. patients according to ge nder % Patie nts No. Gender 64.8 46 Male 35.2 25 Female 100 71 Total 2009) 4 (22مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد في تشخیص األطفال المصابین Dot. ELISAتقییم اختبار االلیزا النقطي الحشائیة اانیابالالشم عبد علي زنجیل جبارة الساعدي رنا صاحب السعدي و ،یدة رشید عليحو ابن الهیثم ، جامعة بغداد -ربیةقسم علوم الحیاة، كلیة الت الخالصة Dotاختیار االلیزا النقطي فائدةملتقیی مرة في العراق ألول ةالدراسهذه أجریت .ELISAللكشف عن أضداد Leishmania donovani. الالشمانیا االحشائیة بمرضإصابتهمالمتوقعة سنوات 8بعمر اقل من "طفال 73 ل V. L. .V.L ریغ أخرى بأمراض لمصابین مصال 57 و ،أصحاء ألشخاصمصال 60 و (Amoebiasis, Giardiasis, T oxop lasmosis ,Sch istosomiasis , Hy datidosis, Ascaiasis , Lupus Erythromatosus , Viral Hep atitis , and Cutaneous Leishmaniasis). ر ، من اإلصابة في مجموعة اإلناثىأوضحت النتائج إن اإلصابة في مجموعة الذكور كانت اعل ٕ وان نسبة انتشا داللة معنویة عالیة بالمقارنة مع الفئات ي سنة وبفارق ذ4.9 - 3 2.9 - 1على ضمن الفئات العمریة المرض كانت األ متوقعة إصابتهم )73 من ( عینة 71 في Leishmania donovani أضدادوجدت .) P>0.01(العمریة األخرى . ولم نجد هذا مع األمراض األخرى لجهازياالحمراري ا الذئبة مرضى داء وجد مع أمصال التفاعل تصالبي .بالمرض %96.3 خصوصیةال و ،%97.3 االختبار بلغت حساسیة 97.4 و %96 القیم التنبوئیة االیجابیة والسلبیة لالختبار . . على التوالي% Dotاختبار . ELISA نوصي باستعماله في ونظرا لذلكحساسیة وخصوصیة وذو ، رخیص الثمن، سریع ، بسیط .اللشمانیا االحشائیةبداء المصليالتشخیص