rK39 ELISA Research Articles

Evaluation of Direct Agglutination Test, rk39 Test, and ELISA for the Diagnosis of Visceral Leishmaniasis

The study reports an evaluation of the direct agglutination test (DAT) with use of promastigote/amastigote antigen, rk39 strip test, and ELISA for diagnosis of visceral leishmaniasis (VL). Out of 94 clinically suspected VL patients, 16 (17%) were seropositive by all the techniques; in addition, 6 were positive in rk39 strip test and ELISA. On retrospective analysis, out of 16 positive by all the techniques, 11 (69%) had demonstrable Leishmania donovani (LD) bodies in their bone marrow samples, while in 5 bone marrow was not examined. Out of 6 that were positive by ELISA and rk39 strip test, 2 had myelofibrosis and 4 had chronic myeloid leukemia. On the basis of bone marrow aspirate positivity, the sensitivity and specificity of DAT were 100% while those of rk39 strip test and ELISA were 100% and 87%, respectively. The study suggests that DAT appears to be the best technique for the serodiagnosis of VL.

Canine visceral leishmaniasis: Performance of a rapid diagnostic test (Kalazar Detect™) in dogs with and without signs of the disease

Current visceral leishmaniasis (VL) control programs in Brazil include the infected dog elimination but, despite this strategy, the incidence of human VL is still increasing. One of the reasons is the long delay between sample collection, analysis, control implementation and the low sensitivity of diagnostic tests. Due to the high prevalence of asymptomatic dogs, the diagnosis of these animals is important considering their vector infection capacity. Hence, a rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. In this study we evaluated the performance of rK39 antigen in an immunochromatographic format to detect symptomatic and asymptomatic Leishmania chagasi infection in dogs and compared the results with those using a crude antigen ELISA. The sensitivity of rK39 dipstick and ELISA were 83% vs. 95%, respectively, while the specificity was both 100%. Our results also demonstrated that the dipstick test was able to detect infected dogs presenting different clinical forms.

Comparative evaluation of direct agglutination test, rK39 and soluble antigen ELISA and IFAT for the diagnosis of visceral leishmaniasis

Five serological tests for the diagnosis of visceral leishmaniasis (VL) were compared: a direct agglutination test (DAT) based on freeze-dried antigen (DAT-fd); a locally produced DAT (DAT-LPC); an IgG ELISA against rK39 (ELISA-rK39); an IgG ELISA for Leishmania chagasi (ELISA-L. chagasi); and an IgG IFAT against L. chagasi. Serum samples from 88 patients with VL, 20 non-infected individuals and 85 patients with others infectious diseases were evaluated. The sensitivity rates were: DAT-fd, 96.6%; DAT-LPC, 95.5%; ELISA-rK39, 88.6%; ELISA-L. chagasi, 89.8%; and IFAT, 92.0% (P>0.05). The specificity for the control groups varied from 53.3% to 100%. DAT-fd had the highest efficiency (97.4%), followed by DAT-LPC (91.7%) and ELISA-rK39 (90.7%). Our data suggest that DAT-fd, DAT-LPC and ELISA-rK39 are useful tests for the diagnosis of VL and could replace IFAT as the routine diagnostic test in Brazil.

Sero-epidemological Survey on Canine Visceral Leishmaniasis and the Distribution of Sandfly Vectors in Northwestern Turkey: Prevention Strategies for Childhood Visceral Leishmaniasis

Visceral leishmaniasis (VL) caused by Leishmania infantum, is an endemic disease in Aegean and Mediterranean Regions among humans and dogs. In this study, a sero-epidemiological survey for VL and cutaneous leishmaniasis (CL), which both are sporadically reported in the region, were carried out in the villages of Eskisehir, Afyon, and Bilecik cities. The study was designed according to the location of the sporadic cases of VL and CL, and blood samples of 111 dogs were randomly collected. Lymph node aspiration samples were taken from dogs that have popliteal lymphadenopathy. Sand flies were also collected using CDC light traps in the several localities. The sera samples were screened using IFAT, ELISA, rk39 ELISA and dip-stick tests for anti-Leishmania antibodies. A total of 15 (13.51 per cent) dogs out of 111 were found to be seropositive by at least one of the tests. The seropositivity ratios among dogs were found to be 27.5 per cent (8/29), 9.09 per cent (4/44) and 7.8 per cent (3/38) in Afyon, Bilecik and Eskisehir cities respectively. Leishmania amastigotes were detected in 4 of the 14 lymph node aspiration samples (eight seronegative, six seropositive), and all of them were seropositive dogs. One year later, two of the dogs were found to be dead and the other two were severely ill. Among the 179 collected Phlebotomus specimens from, Phlebotomus major was found to be abundant (35.7 per cent) and the other species were P. simici (28.5 per cent), P. similis (34.7 per cent) and P. alexandri (1.1 per cent). In the study area, canine VL is more spread than human VL. Because dogs are playing an important role for VL in Mediterranean Basin, and development of appropriate control measures will be necessary for childhood VL.

Evaluation of Enzyme-Linked Immunosorbent Assays, an Immunofluorescent-Antibody Test, and Two Rapid Tests (Immunochromatographic-Dipstick and Gel Tests) for Serological Diagnosis of Symptomatic and Asymptomatic Leishmania Infections in Dogs

Enzyme-linked immunosorbent assays (ELISAs) based on soluble antigens derived from promastigote or amastigote-like stages of Leishmania infantum and on the recombinant rK39 antigen, each in combination with different conjugates [anti-immunoglobulin G1 [IgG1], anti-IgG2, anti-IgG(gamma), and anti-IgG heavy plus light chains], were compared to an immunofluorescent-antibody test (IFAT) and two commercially available rapid test systems (DiaMed-Vet-IT Leish and ID-PaGIA canine leishmaniasis antibody test) for the detection of specific anti-Leishmania antibodies in symptomatic and asymptomatic dogs with proven L. infantum infections. ELISAs based on soluble promastigote and amastigote antigens had very high sensitivities in symptomatic (n = 30; 100%) and asymptomatic dogs (n = 17; 94.1 to 100%), except when combined with the anti-IgG1 conjugate (41.2 to 82.4%). Specificities were high for all combinations (n = 50; 96 to 100%). The rK39 ELISA detected fewer asymptomatic cases (sensitivities, 52.9 to 64.7%) but was highly specific (96 to 100%). The IFAT was 90% sensitive in symptomatic dogs but was significantly less sensitive in asymptomatic cases (29.4%). However, it had an excellent specificity (100%). Test performances of the rapid tests based on the rK39 antigen were comparable to the ELISAs based on the same antigen. ELISAs based on soluble promastigote or amastigote antigens seem to be most suited for the serological diagnosis of canine Leishmania infections in both symptomatic and asymptomatic dogs. IFAT and the rK39 ELISA lack sensitivity in asymptomatic cases but are highly specific. Rapid tests like the rK39 dipstick test or the ID-PaGIA are helpful for confirming clinically suspected cases because of their high specificities in symptomatic animals.

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.

Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey.

Infantile Mediterranean visceral leishmaniasis (IVL) and anthroponotic cutaneous leishmaniasis (ACL) have long been known to exist in the western and southeastern Turkey, respectively. To further study these and other related diseases, a recombinant antigen (rK39) specific to VL was used in an ELISA for serodiagnosis of selected patients and for screening dog reservoir populations in several endemic sites. Among 24 confirmed VL cases from western Turkey, the rK39 ELISA proved to be more sensitive than a combination of cultivation and microscopy of bone marrow aspirates. The specificity of rK39 for leishmaniasis was demonstrated by its lack of cross-reactivity with sera from other human diseases in the same sites. Interestingly, six of the 83 parasitologically proven ACL cases from southeast Turkey were also rK39 positive. The end point titers of the positive VL and CL cases vary from 10(-2) to 10(-5) and from 10(-2) to 10(-3), respectively. The rK39 ELISA was also used to screen 494 apparently healthy dogs from Urfa in southeast Turkey, Manisa/Alasehir near the Aegean Sea, and Karabuk near the Black Sea. Eighteen rK39-positive cases (3.6%), all from the latter two areas, were found to have varying endpoint titers (10(-2)-10(-4)). The high titers predicted increased severity and frequency of the clinical symptoms (i.e., lymphadenopathy, depilation, skin lesion, weight loss and/or death), which were manifested subsequently in 16 of these 18 cases. In addition, more positive canine cases were diagnosed by the rK39 ELISA preclinically than the procedures to detect parasites postsymptomatically in the lymph node aspirates. The use of the rK39 ELISA as a sensitive tool makes it possible to demonstrate coendemicity of canine and human VL, as expected in the case of IVL. The results also point to the possible presence of additional VL types in western Turkey and cutanovisceral type in the southeast part of this country.

A cloned antigen (recombinant K39) of Leishmania chagasi diagnostic for visceral leishmaniasis in human immunodeficiency virus type 1 patients and a prognostic indicator for monitoring patients undergoing drug therapy.

Serologic assays using crude antigens for the diagnosis of visceral leishmaniasis in human immunodeficiency virus type 1 (HIV)-seropositive patients have been shown to lack sensitivity and specificity, particularly in AIDS patients. Antibodies to a cloned antigen, recombinant (r) K39, of Leishmania chagasi are specific for members of the Leishmania donovani complex and have been shown to indicate active disease in immunocompetent persons. This study demonstrated that antibodies to rK39 were also detectable in HIV-seropositive patients coinfected with Leishmania infantum. Furthermore, the rK39 ELISA was more sensitive than an IFA for detecting L. infantum infections in patients with AIDS. In addition, antibody titers to rK39 in HIV-negative patients infected with L. infantum or L. chagasi declined during treatment with meglumine antimoniate or liposomal amphotericin B. In contrast, most patients who clinically relapsed showed increased antibody titers to rK39. These data demonstrate the diagnostic and prognostic utility of rK39 in detecting active visceral leishmaniasis.