Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs

A direst agglutination test (DAT) and an immunofluorescence (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrabida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres ò1:320 and ò1:128). Of 34 samples collected form apparently healthly dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25 per cent. Considering these and previous studies in the adjancent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be high magnitude.

Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs.

A direct agglutination test (DAT) and an immunofluorescence technique (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrábida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres > or = 1:320 and > or = 1:128). Of 34 samples collected from apparently healthy dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25%. Considering these and previous studies in the adjacent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be of high magnitude.

Reactivity of various leishmanial antigens in a direct agglutination test and their value in differentiating post-kala azar dermal leishmaniasis from leprosy and other skin conditions.

A direct agglutination test (DAT) for the detection of post-kala azar dermal leishmaniasis (PKDL) was evaluated in conditions that simulate the disease clinically or immunologically. A reference strain of Leishmania donovani (LEM 1399), and antigen preparations from Leishmania isolates from Bangladeshi patients with post-kala azar dermal leishmaniasis or visceral leishmaniasis were used. A titre of at least 51,200 was obtained in tests of patients with PKDL with all three antigens, whereas a maximum titre of 1600 was recorded in patients with cutaneous leishmaniasis, mucocutaneous leishmaniasis or leprosy. Antigens from dermal isolates of L. tropica (LV 140) and L. braziliensis (LV 65) yielded titres of 1600-6400 in patients with PKDL. The lowest titre recorded in 70 patients tested with the homologous PKDL antigen was 409,600. In patients with leprosy, cutaneous leishmaniasis, syphilis, onchocerciasis, tuberculosis, blastomycosis or vitiligo, titres ranged from 100 to 1600. Tha DAT is better than current parasitological and histopathological methods for the diagnosis of PKDL in areas in which leprosy is co-endemic.

Evaluation of cleaving agents other than trypsin in direct agglutination test for further improving diagnosis of visceral leishmaniasis.

Trypsin treatment of Leishmania promastigote antigen has proved to be indispensible in the direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). In the present study four antigen batches were prepared with pronase (400 micrograms/ml), lipase (0.45% [wt/vol]), pancreatin (0.3% [wt/vol]), or 2-mercaptoethanol (2-ME) (1.2% [vol/vol]) at a ratio of 20:1 versus promastigote packed cell volume or a density of 10(8)/ml. Batches prepared in this way performed satisfactorily when compared with the performance of the initial trypsinated antigen. Even higher was the sensitivity and specificity of the 2-ME-processed antigen, scoring a minimum DAT titer of 1:102,400 in the VL and CVL group and a maximum of 1:400 in the negative control group. Corresponding titers ranging from 1:6,400 to 1:12,800 and 1:800 to 1:1,600 were obtained with the antigen variants processed with pronase, lipase, pancreatin, or trypsin. By combining the use of indigenous Leishmania donovani subspecies from Sudan, Bangladesh, or Morocco and incorporating 2-ME instead of trypsin in the antigen processing step, a threefold increase in titer was attained in sera from the respective areas where VL is endemic. 2-ME-processed antigen suspensions maintained stability at 4 degrees C for up to 9 months, as evidenced by the absence of autoagglutination and the reproducibility of DAT readings with standard sera. The specificity of DAT was further improved by supplementation of the sample diluent with 0.03 M urea and incubation of the test plates at 37 degrees C for 1 h. Titers ranging from 1:200 to 1:12,800 in the sera of patients and laboratory animals infected with various trypanosoma species were significantly reduce (/=1:51,200) against 2-ME-processed antigen, despite the incorporation of urea into the DAT.

Evora district as a new focus for canine leishmaniasis in Portugal.

On the basis of information acquired from local health authorities in Evora district of Portugal on cases of visceral leishmaniasis (VL), an epidemiology survey study was conducted. To determine the prevalence of anti-Leishmania antibodies in the local human and canine populations residing in Evora town and 14 adjacent villages, blood samples collected from 885 children and 3,614 dogs were tested in a direct agglutination test (DAT). Seropositivity for Leishmania parasite obtained by DAT in both endemic populations was further confirmed by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence test (IFAT). For identification of the responsible sandfly vector, 79 biotopes within the study areas were surveyed. In the infantile population assessed, none of the children screened showed an antibody level indicative (titer, > = 1:3200) of visceral leishmaniasis in the DAT. However, agglutinating antibody rates ranging from 0.7% to 6.9% were obtained in dogs residing in Evora and 11 adjacent villages. Concordant seropositivity of 94.04% was obtained by ELISA and IFAT in the same canine population (141) identified by DAT. Of the 159 sandflies captured, 67 were identified as Phlebotomus sergenti; 15, as P. ariasi; 58, as P. perniciosus; and 19, as Sergentomyia minuta. Unlike the results previously reported in Alto-Douro and Algarve districts of Portugal, as compared with the other three species, P. sergenti appears to be more abundant in Evora district.(ABSTRACT TRUNCATED AT 250 WORDS)

Applicability of direct agglutination test (DAT) at a rural health setting in Bangladesh and feasibility of local antigen production.

As part of a large-scale sero-epidemiological survey on visceral leishmaniasis (VL) carried out in Mymensingh district of Bangladesh, applicability of DAT was assessed at the level of a rural health setting in Trishal (upazila) subdistrict. Despite the relatively less optimal conditions encountered, 5854 inhabitants from 7 villages appendant to Trishal were assessed for VL. The demographic distribution for sero-positivity obtained at the rural setting was comparable to that found by DAT as executed at the central laboratory (IEDC&R, Dhaka) on 9619 inhabitants from the same upazila. The overall sero-prevalence rate was 4.4% compared to 3.7% obtained in the population assessed at the central laboratory. In either study, similar VL prevalence rates of 2.1% were obtained in the male populations. Irrespective of sex, younger population (< 20 years) in both studies appeared to have higher VL incidence rate (2.3% and 2.6%) than others of 21- > or = 90 years (1.4% and 1.8%). Local production of DAT antigen employing an authochtonus L. donovani isolate was attempted at the central laboratory (IEDC&R) in Dhaka. By comparison with the reference antigen, titres obtained in all 33 VL sera tested were equally higher (1:6400- > or =: 51200) than in 35 out of 38 negative controls (< or = 1:400-1:1600). A comparable level of reactivity was also obtained in 53 VL and 52 negative control sera using a well characterized L. donovani strain (MHOM/IN/80/D88) from India. However, unlike the reference strain, titres obtained in 7 endemic controls were significantly higher with the authochtonous and homologous antigen (1:3200 - 1:6400) than with the reference (1:100 - 1:1600). The results signify the advantage of employing indigenous L. donovani isolates to further improve DAT sensitivity for detection of early and sub-clinical VL.

[Canine leishmaniasis in the region of Enfidha (Central Tunisia). Assessment of seroprevalence with direct agglutination (DAT) and indirect immunofluorescence (IFAT)]. / La leishmaniose canine dans la région d'Enfidha (Tunisie centrale). Estimation de la séro-prévalence par agglutination directe (DAT) et immunofluorescence indirecte (IFAT).

In order to assess canine leishmaniasis prevalence rate in Enfidha area, considered to be the most important kala-azar focus in Sousse Governorate, a serological survey was carried out in 6 localities. 265 sera were examined by DAT and IFAT. 16 (6.03%) showed positive results for anti-leishmania antibodies with significant variations according to the locality. A fairly DAT-IFAT good correlation was observed.

A killing disease epidemic among displaced Sudanese population identified as visceral leishmaniasis.

A fatal disease epidemic affected the Bentiu area in southern Sudan and led to a mass migration of the Nuer tribe searching for treatment. The initially available information revealed a high mortality rate due to a possible occurrence of tuberculosis, malaria, enteric fever or visceral leishmaniasis (VL). Serological screening of 53 of the most severely affected patients in an enzyme-linked immunosorbent assay (ELISA) or an improved direct agglutination test (DAT) revealed positivity for VL. In 39 of those patients, diagnosis was confirmed by identification of Leishmania donovani amastigotes in lymph node or bone-marrow aspirates. In a total of 2714 patients observed, 1195 (44.0%) had clinical symptoms suggesting VL: DAT positive titers (1:3200-greater than or equal to 1:12800) were obtained in 654 (24.1%), of whom 325 were confirmed parasitologically. Forty-two VL cases died before or during treatment, giving a mortality rate of 6.4%. Among the intercurrent infections diagnosed in the VL population (654), respiratory involvements (31.7%) and malaria (10.7%) were most prevalent. With the exception of four (0.6%), all other VL patients (509) responded readily to sodium stibogluconate. The factors initiating the outbreak are discussed. Malnutrition and nomadic movements to potential VL endemic areas appeared to be the most important. HIV infection as a possible predisposition seemed remote considering the clinical and epidemiological similarity to VL occurring in East Africa, adequate humoral response in DAT, and immediate positive response to specific anti-Leishmania chemotherapy.