Zahra Rezaei; Gholamreza Pouladfar; Amin Ramezani; Zohreh Mostafavi-Pour; Amin Abbasian; Bahador Sarkari; Bahman Pourabbas
Abstract
Background: Visceral leishmaniasis (VL) can lead to death in more than 95% of cases if left untreated. Accurate and early diagnosis has an important role in reducing mortality rate of this disease. Objective: To express recombinant H2B antigen from an Iranian isolate of Leishmania Infantum and evaluate ...
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Background: Visceral leishmaniasis (VL) can lead to death in more than 95% of cases if left untreated. Accurate and early diagnosis has an important role in reducing mortality rate of this disease. Objective: To express recombinant H2B antigen from an Iranian isolate of Leishmania Infantum and evaluate its efficacy in the diagnosis of VL. Methods: The recombinant H2B antigen was produced in a prokaryotic system, and its efficacy for VL diagnosis was evaluated by ELISA. The serum samples from 80 VL patients, 100 individuals from endemic and non-endemic regions of VL, and 58 non-VL patients were collected. VL cases were confirmed based on the clinical sign, positive IFAT (>64), real time PCR, and response to treatment. Results: The H2B gene sequence of the Iranian L. infantum isolate had about 4% diversity in comparison with the H2B gene of the L. infantum counterpart. ELISA, using the produced H2B recombinant antigen, showed sensitivity of 71.25% (95% CI: 60.05%-80.82%) and specificity of 69.62% (95% CI: 61.81%-76.68%) regarding VL diagnosis. Conclusion: Recombinant H2B antigen expressed in the prokaryotic system had suboptimal performance for the serological diagnosis of VL. It seems that the production and expression of recombinant H2B antigen in a eukaryotic system may enhance the performance of this antigen in the diagnosis of VL in Iran.
Behnam Mohammadi-Ghalehbin; Gholam Reza Hatam; Bahador Sarkari; Mehdi Mohebali; Zabih Zarei; Mansoureh Jaberipour; Shahab Bohlouli
Volume 8, Issue 4 , December 2011, , Pages 244-250
Abstract
Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum in Mediterranean basin and is an endemic disease in some parts of Iran. Canines are the main reservoirs of VL in most of the endemic areas. Different serological methods have been introduced for diagnosis of canine visceral leishmaniasis ...
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Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum in Mediterranean basin and is an endemic disease in some parts of Iran. Canines are the main reservoirs of VL in most of the endemic areas. Different serological methods have been introduced for diagnosis of canine visceral leishmaniasis (CVL). Objective: In this survey a Fucose-Mannose Ligand (FML) ELISA, using native L. infantum antigen, was developed and its validity for detection of infected dogs in comparison with direct agglutination test (DAT) and PCR was evaluated. Methods: Blood samples of sixty ownership dogs (≤ 3 years old) were collected from Meshkin-shahr district in Ardabil province, North-west of Iran. Sera were separated for serological assays (DAT and FMLELISA) and the buffy coats were collected for molecular evaluation. Results: Two out of the 60 (3.33%) samples were found to be positive (antibody titer of ≥ 1/320) in DAT while seven of the 60 (11.66%) samples were positive by FML-ELISA. Nine out of 60 (15%) buffy coat samples showed a band about 680 bp indicative of L. infantum in PCR. Three out of 60 dogs had Kala-azar symptoms and were positive by PCR and FML-ELISA, while two of these three dogs had antibody titers >1/320 in their serum samples. The sensitivity and specificity of FML-ELISA for the detection of CVL in both symptomatic and asymptomatic dogs were found to be 77.8% and 100%, respectively. Conclusion: Considering the acceptable sensitivity and high specificity of FMLELISA, use of this serological method can be recommended for epidemiological surveys of CVL.
Elfadil Abass; Abdelhafeiz Mahamoud; Durria Mansur; Mehdi Mohebali; Abdollah el Harith
Volume 8, Issue 3 , September 2011, , Pages 150-158
Abstract
Background: A β-mercaptoethnol (β-ME)-treated promastigote antigen of L. donovani was successfully employed in direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL). Objective: The β-ME-treated antigen was further incorporated into an enzyme-linked immunosorbent ...
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Background: A β-mercaptoethnol (β-ME)-treated promastigote antigen of L. donovani was successfully employed in direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL). Objective: The β-ME-treated antigen was further incorporated into an enzyme-linked immunosorbent assay set-up (β-ME ELISA) and evaluated for VL diagnosis against outcome of reference freeze-dried DAT (FD-DAT) and rK39 strip test (RKT) commercial kits. Methods: Two-hundred and ninety-two sera from patients with high VL suspicion of whom 105 had confirmed L. donovani infection were tested. Results: Relatively higher sensitivities of 93.3% (95% CI: 88.4- 98.2) and 92.4% (95% CI: 87.3-97.5) were determined for β-ME ELISA and FD-DAT as compared to 83.8% (95% CI: 76.7-90.8) for RKT. Of 73 VL sera that scored maximum absorbance values (>0.81) in β-ME ELISA, 66 (90.4%) tested at the highest agglutination titres (>1:51200) in FD-DAT as did 56 (76.7%) also at comparable reaction intensities (3 + colour intensity) in RKT. Compared with FD-DAT (94.7%, 95% CI: 91.5-97.9) or RKT (93.0%, 95% CI: 89.3-96.6), lower specificity was estimated for β-ME ELISA (90.4%, 95% CI: 86.1-94.6). Based both on positive and negative microscopy for L. donovani in organ aspirates of all VL suspects enrolled (292), significantly higher correlation (p<0.01, 0.919) was established between β-ME ELISA and FD-DAT than between β-ME ELISA and RKT (p<0.01, 0.824). Taking into calculation the combined estimates of sensitivity, specificity, positive and negative predictive values, higher agreement (94.8%) was determined between total performance of β-ME ELISA and FD-DAT than between that of β-ME ELISA and RKT (90.7%). Conclusion: Based on results and merits discussed, we recommend application of this β-ME ELISA both for diagnosis of VL at laboratory level and confirmation of results obtained with DAT or RKT in the field.
Mohammad Amin Ghatei; Gholamreza Hatam; Seyed Mohammad Hossein Hosseini; Bahador Sarkari
Volume 6, Issue 4 , December 2009, , Pages 202-207
Abstract
Background: Visceral leishmaniasis (VL) is an endemic disease in some parts of Iran. Many techniques have been used for diagnosis of VL, among which the urine based la-tex agglutination test (KAtex) is a promising one. Objective: To compare three diag-nostic tests of VL including KAtex, ELISA and Direct ...
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Background: Visceral leishmaniasis (VL) is an endemic disease in some parts of Iran. Many techniques have been used for diagnosis of VL, among which the urine based la-tex agglutination test (KAtex) is a promising one. Objective: To compare three diag-nostic tests of VL including KAtex, ELISA and Direct Agglutination Test (DAT) in VL patients and healthy controls in the south west of Iran. Methods: Serum (n = 29) and urine samples (n = 31) were collected from parasitologically confirmed VL patients. Control samples were obtained from healthy individuals (n = 61) and also from patients with infectious diseases other than VL. The collected serum samples were tested by DAT and ELISA using crude antigen from promastigotes of Leishmania infantum and the urine samples were tested by KAtex. Results: Sensitivity and specificity of KAtex for diagnosis of VL was found to be 83.9% and 100%, respectively. Sensitivities of DAT and ELISA were 93.1% and 86.2% and their specificities were 100% and 90.5%, respectively. Conclusion: KAtex yielded a satisfactory sensitivity and specificity in di-agnosis of VL in Iran and can be recommended as a rapid, field applicable and reliable test for diagnosis of VL in this region.
Durria Mansour; Elfadil M. Abass; Abdelhafeiz Mahamoud; Abdallah el Harith
Volume 6, Issue 4 , December 2009, , Pages 208-215
Abstract
Background: Until now, the comparison of the rK39 strip test (RKT) and direct ag-glutination test (DAT) for detection of visceral leishmaniasis (VL) is exclusively based on either positive or negative qualification of the reaction outcome. Objective: In this study, we compared the diagnostic performance ...
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Background: Until now, the comparison of the rK39 strip test (RKT) and direct ag-glutination test (DAT) for detection of visceral leishmaniasis (VL) is exclusively based on either positive or negative qualification of the reaction outcome. Objective: In this study, we compared the diagnostic performance of RKT and DAT for VL both qualitatively and semi-quantitatively. Methods: For comparison based on semi-quantitative grounds, the execution of RKT and DAT was according to the standard procedures. For comparison on semi-qualitative grounds with DAT, the RKT was ap-plied to aliquots from positive samples that were two-fold serially diluted in saline to determine, as for the DAT, the end-point reaction in RKT. Results: While qualita-tively both RKT and DAT demonstrated comparable reliability for VL detection (sen-sitivity = 96% and specificity = 98.7% or 99.3%), no significant correlation (r = 0.13) could be established between intensities of their positive reactions in 25 cases studied. A negative correlation was further determined in those 25 VL cases between the posi-tive intensities of the RKT and antibody levels measured semi-quantitatively with the same procedure (r = -0.36) or the DAT (r = -0.30). Irrespective of the low, moderate or high antibody levels measured with RKT (<1:8 and 1:16-1:32 >1:256) or DAT (< 1:25,600 and 1:51,200- 1:409,600 > 1:3,276,800) in patients with confirmed or uncon-firmed VL infection, exclusively strong positive intensities were obtained with RKT. Conclusion: For further optimizing diagnosis and simultaneously assessing magni-tude of immune response to L. donovani infection in Sudanese patients, the combined application of RKT and DAT is recommended.
Fattaneh Mikaeili; Mahdi Fakhar; Bahador Sarkari; Mohammad H. Motazedian; Gholamreza Hatam
Volume 4, Issue 2 , June 2007, , Pages 116-121
Abstract
Background: The causative agent of visceral leishmaniasis (VL) in Iran is Leishmania infantum (L. infantum) (Mediterranean type) and its major reservoir host is the dog. Ob-jective: To compare the serological methods including direct agglutination test (DAT), indirect immunofluorescent-antibody test ...
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Background: The causative agent of visceral leishmaniasis (VL) in Iran is Leishmania infantum (L. infantum) (Mediterranean type) and its major reservoir host is the dog. Ob-jective: To compare the serological methods including direct agglutination test (DAT), indirect immunofluorescent-antibody test (IFA) and enzyme-linked immunosorbent as-say (ELISA) for serodiagnosis of endemic strain of L. infantum. Methods: 61 blood samples from VL patients referred to Shiraz hospitals and 49 blood samples from con-trol group were collected. Native strain of the parasite isolated from a VL patient from the region was cultured and characterized. Antigens from this L. infantum parasite were used in ELISA and IFA system. Results: Anti-Leishmania antibody was detected in 43 (70.5%), 49 (80.3%) and 51(83.6%) cases using DAT, IFA and ELISA, respectively. Based on these results, sensitivity and specificity of DAT was found to be 70.5% and 100%, respectively. Sensitivities of IFA and ELISA in diagnosis of VL were 80.3% and 83.6% and their specificity was 90.5%. Conclusion: Results of this study showed that DAT and ELISA have the highest specificity and sensitivity in diagnosis of VL. DAT is a simple, cost-effective and field applicable test. Thus, it can be recommended for early and accurate diagnosis of VL, especially in regions where malaria, brucellosis and tu-berculosis are prevalent.