ABSTRACT
ABSTRACT Background: Leishmaniasis is a vector-borne disease caused by a parasite protozoon from the genus Leishmania. Among the molecular techniques applied for detecting these parasites, real-time PCR with High Resolution Melting (PCR-HRM) proved advantageous since it simultaneously determines both the presence and species of the pathogen in one step, through amplification and later analysis of curves generated by melting temperature. Methods: Based on this molecular technique, the goal of this study was to estimate the PCR-HRM sensitivity for Leishmania spp. detection in different canine tissues by evaluating biological samples obtained from popliteal, submandibular, and pre-scapular lymph nodes, from bone marrow and ear pinnae of 28 stray dogs captured in the metropolitan area of Asunción (Paraguay). Results: The rk39 immunochromatographic test showed that 25/28 tested dogs (89%) presented antibodies against L. infantum. In 20/25 dogs that tested positive for rk39 (80%), it was possible to detect Leishmania spp. by PCR-HRM and determine that the species corresponded entirely to L. infantum. Regarding the analysis of different tissues, the parasite was detected in all popliteal lymph node samples, followed by high detection in submandibular (at 95%) and pre-scapular lymph nodes (at 90%), bone marrow (at 85%), and ear pinnae (at 85%). Conclusions: This study demonstrated that the use of real-time PCR-HRM using the molecular marker hsp70 was a highly sensitive method for simultaneously detecting and identifying Leishmania species in different tissues taken from infected dogs. In addition, the usefulness of ear pinnae as easily accessible tissue for molecular diagnosis was emphasized.
ABSTRACT
The aim of the present study was to evaluate the serological methods using ELISA with recombinant-rK39 (ELISA-rK-39) and soluble extract-SE (ELISA-SE) antigens, the indirect fluorescence antibody test (IFAT) in comparison to an immunochromatography rapid diagnostic test (RDT-rK39) and with a direct parasitological exam (PA) for Canine Visceral Leishmaniasis (CVL) diagnosis. The results showed that 89% (60/67) of the dogs were positive for at least one serological diagnostic test. ELISA-SE was the test that detected anti-Leishmania antibodies in the serum of the highest number of dogs (71.6%) followed by ELISA-rK39 (65.7%), IFAT (65.7%) and RDT-rK39 (55.2%). PA detected the lowest numbers (40.3%) of positive dogs. In relation to the total of examined dogs, the Kappa indexes (p ≤ 0.05) showed a good agreement between ELISA-SE and IFAT (88.1%; k = 0.7237), and it was also observed in the comparison of RDT-rK39 with ELISA-SE (83.6%, k= 0.6561), IFAT (83.5%, k= 0.6605) and PA (85.0%, k= 0.7074). A bad agreement was detected in any association of ELISA-rk39 with the other tests in either symptomatic or asymptomatic animals. ELISA as well as RDT using recombinant antigenic protein (rK39) were the methods that detected the lowest prevalence rates (33.3%) of CVL in asymptomatic dogs. In conclusion, only one test does not adequately identify dogs with CVL and it is necessary the association of two or more diagnostic tests. Because of the good agreement indexes of RDT-rK39 when evaluated with ELISA-SE, IFAT and PA it was suggested as a complementary method to be used in association with either ELISA-SE or IFAT, particularly in the symptomatic dogs. Furthermore, new studies are recommended in order to improve the sensitivity of tests mainly for asymptomatic dogs.
O objetivo do presente estudo foi avaliar os métodos sorológicos usando ELISA (Ensaio Imunoenzimático Indireto) com o antígeno recombinante rK39 (ELISA-rK39) e o antígeno extrato solúvel bruto (ELISA-ES) e a RIFI (Reação de Imunofluorescência Indireta) em comparação com o método imunocromatográfico rápido (RDT-rK39) e o parasitológico direto (PA), para o diagnóstico da Leishmaniose Visceral Canina (LVC) em cães de Ilha Solteira, São Paulo, Brasil. Os resultados mostraram que 89% (60/67) dos cães foram positivos por pelo menos um teste diagnóstico sorológico (RIFI, ELISA-ES, ELISA-rk39 ou RDT-rK39) e somente 40,3% (27/67) foram positivos pelo PA. O ELISA-ES foi o teste que detectou anticorpos anti-Leishmania em maior número de cães (71,6%) seguido por ELISA-rK39, RIFI (65,7%) e por RDT-rK39 (55,2%). No total de cães analisados (assintomáticos e sintomáticos), o índice Kappa de concordância (p ≤ 0,05) foi considerado de boa concordância entre ELISA-ES e IFAT (88,1%; k= 0,7237) e entre RDT-rK39 com ELISA-ES (83,6%, k= 0,6561), RIFI (83,5%, k= 0,6605) e PA (85,0%, k= 0,7074). O índice de concordância ruim foi observado em qualquer associação de ELISA-rk39 com todos os outros testes nos animais sintomáticos e nos assintomáticos. Tanto o ELISA como o RDT com proteínas recombinantes (rK39) detectaram a menor porcentagem de cães assintomáticos (33,3%) em relação aos outros testes sorológicos. Em conclusão, somente um método diagnóstico não foi suficiente para identificar todos os cães positivos com LVC, principalmente os assintomáticos e por isso foi necessário a associação de dois ou mais métodos. Em função da boa concordância do teste RDT-rK39 com ELISA-ES, RIFI e PA, o mesmo foi sugerido como um teste complementar ao ELISA-ES ou RIFI para o diagnóstico da LVC, principalmente dos cães sintomáticos. No entanto, novos estudos são recomendados para melhorar a sensibilidade dos testes principalmente para cães assintomáticos.
Subject(s)
Animals , Dogs , Dog Diseases/diagnosis , Dog Diseases/parasitology , Leishmaniasis, Visceral/veterinary , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Leishmaniasis, Visceral/diagnosis , Parasitology/methodsABSTRACT
The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer's instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Young Adult , Antigens, Protozoan , Leishmania braziliensis/immunology , Leishmaniasis, Cutaneous/diagnosis , Protozoan Proteins , Chromatography, Affinity , Leishmaniasis, Cutaneous/immunology , Reagent Kits, Diagnostic , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
O presente estudo teve como objetivo pesquisar a presença de anticorpos anti-Leishmania sp. em equinos provenientes do município de Araçatuba-São Paulo, área endêmica para leishmaniose visceral canina e humana. Dos 466 equinos testados para a presença de IgG anti-Leishmania chagasi pelo método de ELISA, 68 (14,59%) apresentaram sororeatividade, com títulos variando entre 0,324 e 0,813. As amostras de soro dos equinos positivos por ELISA foram testadas também por imunocromatografia, e 19/466 (4,08%) apresentaram resultado positivo. Os resultados obtidos no presente estudo indicaram que esta espécie animal está em contato e deve atrair flebotomíneos transmissores da doença, e salientam a necessidade de investigação mais acurada sobre o real papel dos equinos residentes em áreas endêmicas, de forma a auxiliar nas medidas de controle da doença.
The aim of the present study was to perform a leishmaniasis survey in horses from Araçatuba, São Paulo, an endemic area of Brazil. Of the 466 horses tested for the presence of anti-Leishmania chagasi titers by ELISA, 68 (14.59%) were seropositive, with titers varying between 0.324 and 0.813. ELISA positive samples were also tested by immunocromatography and 19/466 (4.08%) were positive. The results of the present study indicated that equines are in contact and can attract phlebotomines, and highlight the necessity of a more accurate investigation on the role played by the horses living in endemic areas, in order to help to control the spread of the illness.
Subject(s)
Animals , Antibodies/immunology , Horses/classification , Leishmaniasis, Cutaneous/pathology , Enzyme-Linked Immunosorbent AssayABSTRACT
More than 20 million people in Bangladesh are considered at risk of developing visceral leishmaniasis (VL). A community-based active surveillance was conducted in eight randomly selected villages in a highly endemic area of Bangladesh from 2006 to 2008. A total of 6,761 individuals living in 1,550 mud-walled houses were included in the active surveillance. Rapid rK39 dipstick tests were conducted throughout the study period to facilitate the case diagnosis. Individuals with previous or current clinical leishmaniasis were identified on the basis of the case definition of the VL elimination program. Untreated cases of suspected VL were referred to the hospital for treatment. Socioeconomic and environmental information including bed net use was also collected. In 2006, the annual incidence of clinical leishmaniasis in the study area was 141.9 cases per 10,000 population, which was significantly increased by the following year owing to community-based active surveillance for case detection and reporting. However, early case detection and early referral for treatment led to a significant decrease in incidence in 2008. This study suggests that community-based active surveillance using a simple diagnostic tool might play a role in achieving the goal of the VL elimination program.
ABSTRACT
The latex agglutination test (KAtex), direct agglutination test (DAT), and the rK39 immuno-chromatographic strip test (dipstick test) were evaluated for their role in the diagnosis and prognosis of visceral leishmaniasis (kala-azar) in India. Sera and urine samples from 455 subjects—150 confirmed visceral leishmaniasis cases, 160 endemic controls, 100 non-endemic controls, and 45 other febrile diseases—were included in the study. The sensitivity of the KAtex, DAT, and rK39 strip test was 87% [95% confidence interval (CI) 80-96], 93.3% (95% CI 88-100), and 98% (95% CI 93-100) respectively. The specificity of these tests was 98% (95% CI 93-100), 93% (95% CI 87-100), and 89% (95% CI 82-97) for the KAtex, DAT, and rK39 strip test respectively. Fifty cases were followed up and subjected to the KAtex, DAT, and rK39 strip test after 30 days of successful treatment. The DAT and rK39 strip test showed positive results in all the 50 cases whereas the KAtex showed no positive reaction in any case. Based on the results, it is concluded that the sensitivity and specificity of the DAT and rK39 strip test are comparable but the greater convenience of use of the strip test makes it a better tool for the diagnosis of visceral leishmaniasis in the peripheral areas of endemic regions whereas the sensitivity of the KAtex needs to be improved to promote its use as a first-line diagnostic test in the field-setting. It may be used for the prognosis of the disease as antigen becomes undetectable in urine after 30 days of the completion of the treatment. Alternatively, it can be used as an adjunct with rK39 for sero-epidemiological surveys.
ABSTRACT
Un estudio transversal fue desarrollado para evaluar la seroprevalencia de la leishmaniasis visceral zoonótica y la tripanosomiasis americana en 211 caninos mestizos de una zona endémica para los dos protozoarios en el sur del departamento del Tolima. Los anticuerpos séricos anti-Leishmania infantum y anti-Trypanosoma cruzi fueron determinados por medio de las técnicas ELISA y Western Blot, usando los antígenos recombinantes rK39 y TESA, respectivamente. Se realizó xenodiagnóstico a los animales seropositivos a T. cruzi. De acuerdo con los resultados, el 40,28% (85/211) y el 1,42% (3/211) de los caninos presentaron anticuerpos contra L. infantum y T. cruzi, respectivamente. El xenodiagnóstico fue negativo. Los resultados sugieren que los caninos pueden jugar un papel importante como reservorios de L. infantum y fuentes de infección para la población infantil del área de estudio. Sin embargo, los perros podrían tener un papel limitado en la transmisión de T. cruzi. Se hace necesario contar con un algoritmo de pruebas diagnósticas para confirmar el estado real de la infección de los animales y para establecer la presencia de reacciones cruzadas o coinfecciones.
A cross-sectional study was conducted in order to evaluate the seroprevalence of zoonotic visceral Leishmaniasis and American Trypanosomiasis in 211 mongrel dogs from an endemic area for the two protozoans in southern Tolima. Serum anti-Leishmania infantum and anti-Trypanosoma cruzi antibodies were determined by ELISA and Western Blot techniques, using rK39 and TESA recombinant antigens respectively. The Xenodiagnosis was performed on the seropositive to T. cruzi. According to the results, 40.28% (85/211) and 1.42% (3/211) of the dogs tested positive for antibodies against L. infantum and T. cruzi respectively. The Xenodiagnosis was negative. The results suggest that dogs may play an important role as reservoirs of L. infantum and infection sources for children in the study area. However, dogs may play a limited role in the transmission of T. cruzi. It is necessary to have an algorithm for diagnostic tests to confirm the actual status of the animals infection and to establish the presence of cross-reactions or co-infections.
Subject(s)
Antigen-Antibody Complex , Blotting, Western , Enzyme-Linked Immunosorbent Assay , LeishmaniasisABSTRACT
Leishmania infantum es el agente etiológico de la leishmaniasis visceral (LV). Sin embargo, tanto en el Viejo Mundo como en el Nuevo Mundo(América Central), este protozoario ha sido involucrado en casos de leishmaniasis cutánea atípica (LCA). Evidencias clínicas, parasitológicas, moleculares y entomológicas han demostrado que una situación similar está ocurriendo en un área periurbana de Altagracia de Orituco (Guárico, Venezuela). Con la finalidad de contribuir al entendimiento de este nuevo escenario epidemiológico, se realizó un estudio transversal en el cual se determinó la reactividad a la prueba de leishmanina [Leishmanin skin test (LST)] y la seroprevalencia a antígenos crudos de Leishmania spp. y al antígeno específico rK39 mediante la prueba de ELISA en una muestra de ocho casos de LCA detectados en el periodo 1997-2000, en sus cohabitantes (n=15) y en una muestra de 233 de la comunidad igualmente expuestas a riesgo. La dermoprevalencia resultó ser de 31,6% (67/212). El 28,8% (67/233) resultó positivo a la prueba de ELISA usando promastigotas de L. infantum (=L. chagasi) y 13,3% (31/233) poseían anticuerpos anti-L. braziliensis. Así mismo, la prueba de ELISA usando el antígeno rK39 resultó positiva en uno de los ocho casos estudiados y en dos de sus cohabitantes; así como también en 13,7% (32/233) de los individuos residentes en la comunidad. Los resultados obtenidos indican la circulación de especies del subgénero Leishmania y del subgénero Viannia en el área en estudio y que la presencia de anticuerpos anti-Leishmania en general se encontró asociada a la edad y al tiempo de residencia en la zona
Leishmania infantum is the etiological agent of the visceral leishmaniasis (VL). However, in the OldWorld as well in the New World (Central America), thisprotozo an has also been involved in cases of atypicalcutaneous leishmaniasis (ACL). Clinical, molecularand entomological evidences have demonstrated thata similar situation is happening in a peri-urban areaof Altagracia de Orituco (Guárico state, Venezuela). With the purpose of contributing to the understandingof such an epidemiological scenario, a cross-sectionalstudy was carryed out using the leishmanin skin test(LST) and screening for the seroprevalence to crudeantigens of Leishmania spp. and rK39 by means ofthe ELISA test in cases of ACL reported in the period1997-2000 (n=8), among their co-inhabitants (n=15) and in a randomly selected sample of 233 people. Thedermoprevalence was 31.6% (67/212); 28.8% (67/233)were positive to the ELISA test using promastigotes of L. infantum (= L. chagasi) and 13.3% (31/233) hadantibodies anti-L. braziliensis. In addition, the test of ELISA using the antigen rK39 was positive in oneout of the eight cases studied and in two out of theirco-inhabitants; as well as in 13.7% (32/233) of theinhabitants of the community. The results obtained inthis epidemiological study indicate the circulation of species of the sub-genus Leishmania as well as of thesubgenus Viannia. The presence of antibodies anti-Leishmania in general was associated to the age andthe time of residence in the zone
Subject(s)
Humans , Male , Adolescent , Adult , Female , Child , Middle Aged , Enzyme-Linked Immunosorbent Assay , Leishmania infantum/pathogenicity , Leishmaniasis, Visceral/diagnosis , Eukaryota , ParasitologyABSTRACT
O teste imunocromatográfico rápido IT-LEISH® (DiaMed IT-LEISH®) foi validado para o diagnóstico da leishmaniose visceral (LV) em quatro áreas endêmicas do Brasil. O desempenho do IT-LEISH® foi comparado ao da reação de imunofluorescência indireta e da reação imunoenzimática utilizando os antígenos solúveis de Leishmania chagasi e o recombinante K39. Foram incluídos no estudo 332 pacientes com quadro clínico sugestivo de LV, sendo 213 casos de LV confirmados parasitologicamente e 119 não-casos com confirmação de outra etiologia. O teste IT-LEISH® apresentou sensibilidade de 93% e especificidade de 97%. As técnicas RIFI, ELISA L. chagasi e ELISA rK39 apresentaram sensibilidade de 88%, 92% e 97% e especificidades de 81%, 77% e 84%, respectivamente. Os resultados confirmam a validade do teste IT-LEISH® para o diagnóstico da LV no Brasil
Subject(s)
Male , Female , Humans , Child , Adolescent , Adult , Middle Aged , Aged , Hematologic Tests , Leishmania donovani/parasitology , Leishmaniasis, Visceral/diagnosisABSTRACT
O teste imunocromatográfico rápido IT-LEISH® (DiaMed IT-LEISH®) foi validado para o diagnóstico da leishmaniose visceral (LV) em quatro áreas endêmicas do Brasil. O desempenho do IT-LEISH® foi comparado ao da reação de imunofluorescência indireta e da reação imunoenzimática utilizando os antígenos solúveis de Leishmania chagasi e o recombinante K39. Foram incluídos no estudo 332 pacientes com quadro clínico sugestivo de LV, sendo 213 casos de LV confirmados parasitologicamente e 119 não-casos com confirmação de outra etiologia. O teste IT-LEISH® apresentou sensibilidade de 93% e especificidade de 97%. As técnicas RIFI, ELISA L. chagasi e ELISA rK39 apresentaram sensibilidade de 88%, 92% e 97% e especificidades de 81%, 77% e 84%, respectivamente. Os resultados confirmam a validade do teste IT-LEISH® para o diagnóstico da LV no Brasil
Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Leishmania donovani/parasitology , Leishmaniasis, Visceral/diagnosis , Hematologic TestsABSTRACT
En 1999, en el Hospital del Niño se registró un nuevo caso de Leishmaniasis visceral sobre un niño de dos años proveniente del cantón de Taipiplaya (Provincia Caranavi). Por este motivo se realiza en este cantón una evaluación transversal de la leishmaniasis visceral mediante pruebas serológicas y moleculares involucrando a 122 individuos clínicamente sanos y un individuo con infección positiva a Leishmania cutánea, Se demostró la circulación de Leishmania sp. en 32,3% de sujetos estudiados. El 14.4% de la población examinada presentó anticuerpos anti-rk39, demostrándose la circulación de Leishmania chagasi responsable de la leishmaniasis visceral. No podemos descartar la posibilidad de la existencia de coinfecciones mixtas inter-especie de Leishmania como también de coinfecciones mixtas por Leishmania sp. y Trypanosoma cruzi, responsable de la enfermedad de Chagas.
In 1999, in the "Hospital del Niño". a new case of visceral leishmaniasis was identified in a 2 years old child from Taipiplaya in the Caranavi district. For this reason, a visceral leishmaniasis evaluation using serological and molecular tests was realized on 122 healthy people and also on one leishmania cutanea infected person. Leishmania spp was present in 32,3 % of the studied people and 14,4 % had an anti-rk39 antibody, attesting the existence of Leishmania chagasi responsible for visceral leishmaniasis. The possibility of mixed infections with other Leishmania species as well as mixed infections Leishmania spp and Trypanosama cruzi, responsible for chagas disease should not be discarded.
ABSTRACT
Objective To analyze the status of Leishmania infantum asymptomatic infection in human population of a Kala-azar endemic area in Wenxian County,Gansu Province,and to evaluate the tests used.Methods Blood samples were tested by PCR using two pairs of primers,RV1-RV2 and K13A-K13B,for detecting Leishmania-specific DNA.ELISA and rK39-dipstick were used to detect Leishmania-specific antibodies.Results The positive rate of PCR,ELISA and rK39-dipstick was 30.9%(83/269),24.2%(65/269) and 0(0/269) respectively.Conclusion The prevalence of asymptomatic infection of L.infantum in humans is high in the area.PCR test based on RV1-RV2 and K13A-K13B primer pairs is a sensitive and specific method for detecting the asymptomatic infection.