Direct Agglutination Test Titres Research Articles

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

Serological and molecular survey of Leishmania infection in dogs from Luanda, Angola

BackgroundCanine leishmaniosis (CanL) due to Leishmania infantum is a global zoonosis endemic in more than 70 countries in Europe, North Africa, Asia and America; however, data on this infection is scarce from southern Africa. The aim of this study was to survey dogs in Luanda, Angola, for Leishmania infection.FindingsOne hundred-and-three dogs presented to a veterinary medical centre in Luanda were serologically and molecularly assessed for Leishmania with the direct agglutination test (DAT) and polymerase chain reaction (PCR). Two dogs were seropositive, with DAT titres of 800 and ≥6400; the latter was also found to be PCR-positive and confirmed to be infected with L. infantum by DNA sequence analysis. No other dog was found to be PCR-positive. The first dog had been imported from Portugal, but the latter had never left Angola (neither had its parents), strongly suggesting an autochthonous infection.ConclusionsAlthough other cases of CanL have previously been described in the country, this is the first reported study of canine Leishmania infection at the population level, as well as the first report on the molecular characterization of L. infantum in dogs from Angola.

Performance of an indigenous β-mercaptoethanol-modified antigen in comparison with a commercial reference in direct agglutination test for detection of canine visceral leishmaniasis

We compared the performance of a locally produced β-mercaptoethanol-modified promastigote antigen (β-ME-Ag) of an indigenous Leishmania infantum strain against that of a trypsinized Leishmania donovani reference (REF-Ag) in the direct agglutination test (DAT) for detection of canine visceral leishmaniasis (CVL). One hundred and fifty-one serum samples collected from dogs belonging to four groups with different conditions were included. At a DAT titre of 1 : 320, statistically determined as optimal cut-off value for β-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for β-ME-Ag in comparison with 96.6 % and 100 %, respectively, for REF-Ag. Overall, levels of agglutination titres recorded for the two antigens were highly concordant (Cohen's κ = 0.879) in both the CVL and non-CVL groups. Based on current results, and ease experienced in processing the antigen and reading the test outcome, we recommend incorporation of β-ME-Ag in DAT for confirmation or exclusion of suspected CVL in dogs.

Prevalence of antibodies to Leishmania infantum and Toxoplasma gondii in horses from the north of Portugal

BackgroundLeishmania infantum and Toxoplasma gondii are protozoa with zoonotic and economic importance. Prevalences of antibodies to these agents were assessed in 173 horses from the north of Portugal.FindingsAntibodies to L. infantum were detected by the direct agglutination test (DAT); seven (4.0%) horses were seropositive with DAT titres of 200 (n = 5), 800 (n = 1) and ≥ 1600 (n = 1). Antibodies to T. gondii were assayed by the modified agglutination test (MAT); 23 (13.3%) horses were seropositive with MAT titres of 20 (n = 13), 40 (n = 5), 80 (n = 3) and ≥ 160 (n = 2). No statistical differences were found among equine categories of gender (female, male and gelding), age (1.5–6, 7–12 and 13–30 years), type of housing (indoors and mixed/outdoors), ability (recreation, farming and sports) and clinical status (apparently healthy and sick) for both agents.ConclusionsHorses are exposed to and may be infected with L. infantum and T. gondii in the north of Portugal.

Serological markers for Leishmania donovani infection in Nepal: agreement between direct agglutination test and rK39 ELISA

Visceral leishmaniasis (VL) is an important vector-borne disease caused by Leishmania donovani in the Indian subcontinent. The actual incidence and role of asymptomatic infections in the region are not wellknown. We used the direct agglutination test (DAT) and the rK39 ELISA as L. donovani infection markers in 10 VL endemic villages in Nepal. DAT titre distribution showed two subgroups in the population (infected and non-infected individuals), while rK39 did not. The agreement between both tests was moderate (j = 0.53; 95% CI 0.49–0.57). More research is needed to develop validated markers for Leishmania infection.

Longitudinal Seroepidemiologic Study of Visceral Leishmaniasis in Hyperendemic Regions of Bihar, India

We conducted a seroepidemiologic study of visceral leishmaniasis (VL) in hyperendemic communities in Bihar, India, to determine its seroprevalence. A direct agglutination test (DAT) and rK39 antigen strip test were used as serologic tests. Capillary blood samples were collected on filter papers from 870 healthy persons (574 and 296 from households with or without VL, respectively). Of these persons, 230 (26.43%) were positive by DAT (titer > or =1:1,600) and 120 (13.79%) were positive by the rK39 antigen strip test. During a two-year follow-up, 25 persons developed VL; 1 and 8 persons were positive by the rK39 strip test and DAT, respectively, and 1 was positive by both tests. Fifteen (2.57%) persons who were seronegative at baseline also developed VL. Disease occurred more among persons living in the same household (24 of 25). However, there was no significant difference in disease conversion among children (5-15 years of age) and adults (> 15 years of age). Seropositivity among asymptomatic persons is not a predictor for development of VL.

Use of a Newly Developed β-Mercaptoethanol Enzyme-Linked Immunosorbent Assay To Diagnose Visceral Leishmaniasis in Patients in Eastern Sudan

Corroboration of serology results is essential for restricting the risk of inappropriate antileishmanial prescription. A direct agglutination test (DAT) and a recently developed beta-mercaptoethanol-modified enzyme-linked immunosorbent assay (beta-ME ELISA) based on the use of antigen prepared as described for the DAT were applied to 416 sera from two Sudanese populations with and without clinical evidence of visceral leishmaniasis (VL). Of 285 sera with the lowest antileishmanial DAT titers (</=1:100 to 1:1,600), 270 (94.7%) scored comparable minimum beta-ME ELISA absorbance values (</=0.1 to 0.26). In 117 sera that demonstrated the highest DAT titers (1:12,800 to >/=1:25,600), 86 (73.5%) scored maximum (0.81 to >/=1.35) and 30 (25.6%) medium (0.27 to 0.80) beta-ME ELISA absorbance values. VL diagnosis was established for 142 (44.1%) patients in the VL-symptomatic group (n = 322), based on positive microscopy for Leishmania donovani in lymph node aspirates or positive DAT (titer, >/=1:3,200). Of the 125 sera from the symptomatic patients for whom microscopy was positive for VL, 111 (88.8%) had comparable positive DAT and beta-ME ELISA readings. In all 17 sera from the symptomatic DAT-positive patients for whom leishmaniasis was not established by microscopy but who responded favorably to antileishmanial therapy, absorbance values (>/=0.27) indicative of VL were obtained by beta-ME ELISA. Of 197 symptomatic patients for whom microscopy was negative for VL, 172 (87.3%) tested negative in beta-ME ELISA and 180 (91.4%) in DAT. Based on the high reliability demonstrated here for VL detection, beta-ME ELISA fulfills the requirement of confirming DAT results in patients manifesting suspected VL.

UTILITY OF LYMPH NODE ASPIRATION IN THE DIAGNOSIS OF VISCERAL LEISHMANIASIS IN SUDAN

We evaluated lymph node aspiration (LNA) as a simple diagnostic procedure for visceral leishmaniasis (VL). Lymph node aspiration was compared with the direct agglutination test (DAT) using a diagnostic titer > or = 1:6,400 in 7,880 suspected VL patients in eastern Sudan. Compared with DAT, LNA had a sensitivity of 65.1% (95% confidence interval = 63.5-66.6%). Parasite density in LNA correlated strongly with DAT titers (P < 0.0001), and low parasite density accounted for 78.1% of positive LNA results with DAT titers < 1:6,400 (n = 782). Risk factors predictive of a positive LNA result were an age of 1-29 years, male sex, a hemoglobin level < 10.0 g/dL, a DAT titer > or = 1:800, and a location with a higher prevalence of VL. Lymph node and splenic aspirations were similarly accurate as tests of cure after treatment of 50 VL patients in southern Sudan. Pre-treatment LNA results were negative in 20 cases of severe post kala-azar dermal leishmaniasis.

Anti- Leishmania humoral and cellular immune responses in naturally infected symptomatic and asymptomatic dogs

Canine infections with Leishmania infantum represent a considerable veterinary medical and public health problem. In this study, immunoglobulin G1 (IgG1) and IgG2 specific humoral responses were measured and compared with the delayed type hypersensitivity (DTH) cellular response to a leishmanin, in three groups of dogs clinically and serologically characterised as: (I) asymptomatic and direct agglutination test (DAT)-seronegative; (II) asymptomatic and DAT-seropositive; (III) DAT-seropositive and symptomatic. IgG2 was regarded as a marker of disease, since significantly higher levels of this subclass were recorded in the symptomatic dogs. In contrast, the IgG1 response could not be related to clinically relevant infection. A high correlation was observed between IgG2 level and DAT titre; the correlations between IgG1 and IgG2 levels, and between IgG1 level and DAT titre were lower. This may indicate that IgG2 is the main subclass in the specific humoral response which is detected by the DAT. A reduced IgG2 response, albeit not significantly different, was recorded among dogs with clear cellular immune responses detected by a DTH positive reaction. Furthermore, no correlations were observed between cellular response measured by DTH and humoral responses quantified by DAT titre or IgG1 and IgG2 levels. Combining serology and DTH skin test is a practical procedure to assess anti- Leishmania immune responses in dogs.

Epidemiological study of kala-azar by direct agglutination test in two rural communities of eastern Nepal.

We conducted a sero-epidemiological study of kala-azar in two endemic communities (Kasaini and Gidhaniya) situated in the Terai (plain) of eastern Nepal. Direct agglutination test (DAT) was used as a serological test for screening. Capillary blood samples were collected by filter paper method from 601 (96%) people of a total population of 628 in Kasaini and from 482 (94%) people of 515 in Gidhaniya. Positive DAT titres (1:2000) were found in 66 (6.09%) of 1083 sera tested. The male-female sero-prevalence ratio was 1.44:1 and the age group of 15 years and above was most affected. Among the bone marrow aspirates collected from 66 DAT seropositive cases, only 19 were positive for Leishmania donovani (LD bodies). Of the 47 DAT seropositive but LD bodies' negative cases, three were clinically active cases of kala-azar. Another nine developed clinical symptoms of kala-azar during 6 months follow-up and 23 were cases that had received prior treatment for kala-azar (within 1 year). The results of this study show the potential of the DAT on filter paper as a screening test for the surveillance of kala-azar at a community level.

Evaluation of a standardized direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis in Kenya

A prototype test kit being developed, by the World Health Organization (WHO), for the diagnosis of visceral leishmaniasis (VL) was evaluated in the Baringo district of Rift Valley province in Kenya. The screening of approximately 10,000 individuals for the signs of VL produced 305 suspected cases. These cases and 304 controls matched for sex and age (+/- 2 years) were then tested with the kit, which is based on a direct agglutination test (DAT). The evaluation was a three-stage process. The first stage, the field screening, involved screening filter-paper samples of dried blood from the suspects and controls at a DAT titre of 1:500. The second stage, the laboratory titration, involved screening of the same individuals by testing freshly eluted filter-paper samples at 1:500 to 1:2000 dilution. In the third stage, the full-scale titration, all samples that had been positive at 1:2000 were titrated at 1:500-1:512,000. All the suspects giving DAT titres of 1:2000 or higher were considered positive for VL. This diagnosis was checked, whenever possible, by the examination of smears and/or cultures of splenic aspirates for leishmanial parasites. Those found to be parasitologically positive were put on a standard treatment regime of 20 mg sodium stibogluconate (Pentostam)/kg.day. Although 42 (13.8%) of the 305 clinical suspects investigated were DAT-positive (at 1:2000), it was only possible to take splenic aspirates from 32. Four (12.5%) of these 32 were apparently false-positives by DAT, as no parasites could be detected in their splenic aspirates. The others provided positive smears and cultures (27 cases) or a negative smear but a positive culture (one case). It was possible to re-examine two of the four serologically positive but parasitologically negative VL suspects at a 3-month follow-up: neither had a palpable spleen, one had seroconverted and the other had much lower DAT titre (1:32,000) than when investigated previously (1:128,000). All the parasitologically confirmed cases remained DAT-positive (1:2000) at this follow-up. The low cut-off titre (1:2000) and the simple procedure should make the kit suitable for use by health workers at all levels of primary-health care, including those with limited training and skills, for screening rural communities at risk of VL.

Evaluation of a standardized direct agglutination test (DAT) for the diagnosis of visceral leishmaniasis in Kenya

A prototype test kit being developed, by the World Health Organization (WHO), for the diagnosis of visceral leishmaniasis (VL) was evaluated in the Baringo district of Rift Valley province in Kenya. The screening of approximately 10,000 individuals for the signs of VL produced 305 suspected cases. These cases and 304 controls matched for sex and age (+/- 2 years) were then tested with the kit, which is based on a direct agglutination test (DAT). The evaluation was a three-stage process. The first stage, the field screening, involved screening filter-paper samples of dried blood from the suspects and controls at a DAT titre of 1:500. The second stage, the laboratory titration, involved screening of the same individuals by testing freshly eluted filter-paper samples at 1:500 to 1:2000 dilution. In the third stage, the full-scale titration, all samples that had been positive at 1:2000 were titrated at 1:500-1:512,000. All the suspects giving DAT titres of 1:2000 or higher were considered positive for VL. This diagnosis was checked, whenever possible, by the examination of smears and/or cultures of splenic aspirates for leishmanial parasites. Those found to be parasitologically positive were put on a standard treatment regime of 20 mg sodium stibogluconate (Pentostam)/kg.day. Although 42 (13.8%) of the 305 clinical suspects investigated were DAT-positive (at 1:2000), it was only possible to take splenic aspirates from 32. Four (12.5%) of these 32 were apparently false-positives by DAT, as no parasites could be detected in their splenic aspirates. The others provided positive smears and cultures (27 cases) or a negative smear but a positive culture (one case). It was possible to re-examine two of the four serologically positive but parasitologically negative VL suspects at a 3-month follow-up: neither had a palpable spleen, one had seroconverted and the other had much lower DAT titre (1:32,000) than when investigated previously (1:128,000). All the parasitologically confirmed cases remained DAT-positive (1:2000) at this follow-up. The low cut-off titre (1:2000) and the simple procedure should make the kit suitable for use by health workers at all levels of primary-health care, including those with limited training and skills, for screening rural communities at risk of VL.

Visceral leishmaniasis in Bangladesh: the value of DAT as a diagnostic tool

The direct agglutination test (DAT) was performed on 480 serum samples from suspected cases of visceral leishmaniasis (VL) in different parts of Bangladesh. Significant titres (⩾1:3200) were found in 257 sera (53.5%). All patients with positive bone-marrow aspirates also had significant DAT titres. The male:female seroprevalence ratio was 2:1 and the age-group 0–20 years was the most affected. The DAT proved a simple, economical and reliable diagnostic test for VL.

Evaluation of direct agglutination test (dat) and elisa for serodiagnosis of visceral leishmaniasis in india

The direct agglutination test (DAT) and the enzyme-linked immunosorbent assay (ELISA) were used for serodiagnosis of parasitologically confirmed Indian visceral leishmaniasis (VL) cases. All the sera of VL cases were positive by both the methods. DAT titres of VL cases were greater than or equal to 1:3,200, and ELISA values were greater than or equal to 0.55 1:400 dilution. In the control group, sera of widely prevalent diseases of India, such as leprosy, tuberculosis, malaria, and liver cirrhosis, were included. Both tests could discriminate between VL and other patients of the control group. The sera of post-kala-azar dermal leishmaniasis (PKDL) patients gave OD values of greater than 0:55 and had DAT titres of 1:1,600. Both tests are sensitive and specific for the diagnosis of VL cases. DAT, being simpler and more economical, will be suitable for diagnosis and epidemiological studies for VL under rural conditions of India.

Introduction of an improved direct agglutination test for the detection ofLeishmania infantum infection in Southern France

An improved direct agglutination test (DAT) is assessed for the detection of Leishmania infantum antibodies in man and in the canine reservoir in Southern France. The test performance in 32 human visceral leishmaniasis patients was in agreement with the parasitological diagnosis and the immunofluorescence (IFAT) results. In six patients diagnosed earlier as kala-azar cases, both DAT and IFAT revealed negative results. The specificity of the DAT was reproduced in this study by the absence of cross-reactions with sera from other patients. In the dog reservoir, the DAT results were compared with those obtained by IFAT, the enzyme-linked immunosorbent assay (ELISA), cross-over electrophoresis and the latex agglutination test. All 34 dogs with parasitologically positive lymph node aspirates also had positive DAT titres (less than or equal to 1:640); the sensitivity in the other four tests was relatively low. Positive DAT and IFAT results were found in 16 unconfirmed cases of canine leishmaniasis, most probably due to prepatent L. infantum infections.