Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection.

BACKGROUND: Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE: We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS: Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS: For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS: For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas.

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2-89.7) and 95.6% (88.8-98.6), and specificity (95% CI) was 93.3% (85.9-97.2) and 97.8% (91.8-99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5-93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5-98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5-98.0), rK28-ELISA (95.9%, 95% CI: 90.5-98.5), and rK39-ELISA (94.3%, 95% CI: 88.4-97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9-72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5-99.2), rK28-ELISA (95.2%, 95% CI: 87.9-98.5), and rK39-ELISA (95.2%, 95% CI: 87.9-98.5). There was no difference in sensitivity and specificity across localities. Cross-reactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95-ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Spatial distribution of canine Leishmania infantum infection in a municipality with endemic human leishmaniasis in Eastern Bahia, Brazil.

Efforts to control a zoonotic disease such as visceral leishmaniasis (VL) caused by Leishmania infantum can be successful if they rely on comprehensive data on animal infection. In Bahia state, Brazil, human VL is endemic, yet some areas have no epidemiological data on canine L. infantum infection and canine leishmaniasis (CanL) to date. We aimed to perform an epidemiological study describing the spatial distribution and characterizing canine L. infantum infection in two districts of the municipality of Muritiba, where human cases have occurred. Brazilian official serodiagnostic protocol (ELISA and immunochromatographic tests), PCR and clinical examination were performed in 351 owned dogs. A seroprevalence of 15.7% (55/351) was found, and L. infantum identified in 88.8% (32/36) of PCR tested samples. Spatial distribution of positive dogs indicated infection in both urban and rural districts. There was no association between seropositivity and sex or breed, but dogs older than 2 years were 3.8 times more likely to be seropositive (95% CI 1.57 - 9.18) than younger dogs. Among seropositive dogs, 80% (44/55) had clinical manifestations of CanL: 75% (33/44) presented dermatopathy, 50% (22/44) emaciation, and 29.5% (13/44) ophthalmopathy. This is the first report on canine seroprevalence and natural L. infantum infection in Muritiba, Bahia.

Correction: Performance of rK39-based immunochromatographic rapid diagnostic test for serodiagnosis of visceral leishmaniasis using whole blood, serum and oral fluid.

[This corrects the article DOI: 10.1371/journal.pone.0230610.].

Performance of rK39-based immunochromatographic rapid diagnostic test for serodiagnosis of visceral leishmaniasis using whole blood, serum and oral fluid.

BACKGROUND: The development of rK39-based immunochromatographic rapid diagnostic tests represents an important advance for serodiagnosis of visceral leishmaniasis, being cheap and easy to use at the point of care (POC). Although the use of rK39 have considerably improved the sensitivity and specificity of serological tests compared with total antigens, great variability in sensitivity and specificity was reported. This study aimed at the evaluation of "Kalazar Detect™ Rapid Test, Whole Blood" (Kalazar Detect RDT) for Visceral Leishmaniasis (VL) diagnosis using oral fluid, whole blood and serum specimens collected at different endemic areas of VL of Brazil. METHODOLOGY: To evaluate Kalazar Detect RDT, oral fluid, whole blood and serum specimens from 128 VL patients, 85 healthy individuals, 22 patients with possible cross-reactivity diseases and 20 VL/aids coinfected patients were collected and assayed at the POC. PRINCIPAL FINDINGS AND CONCLUSIONS: The performance of Kalazar Detect RDT in whole blood and serum was similar; however, using oral fluid, the sensitivity was low. Particularly in samples from the city of Natal, Rio Grande do Norte state in Northeastern Brazil, we observed low sensitivity, 80.0% (95% CI: 62.7-90.5), using whole blood and serum, and poor sensitivity, 43.3% (95% CI: 27.4-60.8) with oral fluid. Those values were much lower than in the other regions, where sensitivity ranged from 92.7-96.3% in whole blood and serum, and 80.0-88.9% in oral fluid. Besides, in VL/aids coinfected patients, lower sensitivity was achieved compared with VL patients. In samples from Natal, the sensitivity was 0.0% (95% CI: 0.0-49.0) and 25.0% (95% CI: 4.6-69.9), using oral fluid and serum/whole blood, respectively; in samples from the other regions, the sensitivity ranged from 40.0-63.6% and 80.0-81.8%, respectively. As for specificity, high values were observed across the fluids, 100.0% (95% CI: 96.5-100.0) in whole blood, 96.3% (95% CI: 90.8-98.5) in serum, and 95.3% (95% CI: 89.5-98.0) in oral fluid; across localities, specificity ranged from 85.7-100.0%. Serum samples sent by the collaborating centers to Instituto de Medicina Tropical (n = 250) were tested by Kalazar Detect RDT, Direct Agglutination Test, Indirect immunofluorescence assay, Enzyme-linked immunosorbent assay, and IT-Leish® RDT. The regional difference in the performance of rK39-based RDT and lower sensitivity in Leishmania/HIV coinfected patients raise concern on the routine use of these products for the diagnosis of VL.

Asymptomatic infections in blood donors harbouring Plasmodium: an invisible risk detected by molecular and serological tools.

BACKGROUND: Transfusion-transmitted malaria due to asymptomatic Plasmodium infections is a challenge for blood banks. There is a lack of data on the prevalence of asymptomatic infected blood donors and the incidence of transfusion-transmitted malaria in low endemicity areas worldwide. We estimated the frequency of blood donors harbouring Plasmodium in an area in which asymptomatic infections have been reported. MATERIAL AND METHODS: To estimate the frequency of blood donors harbouring Plasmodium we used microscopy and molecular tools. Serological tests were applied to measure the exposure of candidates to Plasmodium antigens. Venous blood was collected from 91 candidates attending the "Pró-Sangue" Blood Centre Foundation in São Paulo, who lived in the municipality of Juquitiba, São Paulo, Brazil, where sporadic autochthonous cases of malaria have been described. Blood samples were used for parasitological, molecular and serological studies. RESULTS: Among the 91 samples examined, rare Plasmodium forms were observed in two donors. Genus real-time polymerase chain reaction analysis demonstrated Plasmodium amplification in three candidates and species-specific nested polymerase chain reaction identified P. malariae in two. ELISA-IgG was reactive in 42.9% of samples for P. vivax (Pv-MSP119) and in 6.6% for P. falciparum (Pf-Zw). ELISA-IgM was reactive in 2.2% of samples for P. vivax and in 4.4% for P. falciparum. An indirect immunofluorescence assay was reactive for P. malariae in 15.4% of cases. DISCUSSION: Reservoirs of Plasmodium represent a challenge for blood banks, since studies have shown that high levels of submicroscopic infections can occur in low transmission areas. The risk of transfusion-transmitted malaria presented here points to the need to conduct molecular investigations of candidate donors with any positive malarial antibody test.

Leishmania infection in blood donors: A new challenge in leishmaniasis transmission?

Transfusion-transmitted leishmaniasis has been a concern in regions endemic for the disease. Whether immediate or delayed, the risks posed by this mode of transmission call for careful assessment. The purpose of this study was to detect Leishmania infection in blood donors living in an endemic area and to investigate progression to the disease in these individuals. Immunofluorescent antibody test, enzyme-linked immunosorbent assay, leishmaniasis rapid test, and the polymerase chain reaction were applied to 430 donors in an initial evaluation. Of those donors with at least one positive test, 50 were reevaluated four years later by the same methods, as were 25 controls who had been negative on the same tests. In the first evaluation, Leishmania infection was detected in 41.4% (95% CI: 36.7-46.1) of donors (n = 430). None of the 75 reevaluated individuals had developed the disease, but retesting revealed positivity in at least one test in 36.0% (95% CI: 25.1-46.9) of donors. Of the 50 initially testing positive, 50% remained so on retesting. Of the 25 initially negative controls, two tested positive in the subsequent evaluation. The severity of the parasitosis and the risk of transfusion transmission warrant investigation of the potential inclusion of methods for Leishmania detection into blood banks for effective screening of infected donors.

Recombinant protein KR95 as an alternative for serological diagnosis of human visceral leishmaniasis in the Americas

In the Americas, visceral leishmaniasis (VL) is caused by the protozoan Leishmania infantum, leading to death if not promptly diagnosed and treated. In Brazil, the disease reaches all regions, and in 2020, 1,933 VL cases were reported with 9.5% lethality. Thus, an accurate diagnosis is essential to provide the appropriate treatment. Serological VL diagnosis is based mainly on immunochromatographic tests, but their performance may vary by location, and evaluation of diagnostic alternatives is necessary. In this study, we aimed to evaluate the performance of ELISA with the scantily studied recombinant antigens, K18 and KR95, comparing their performance with the already known rK28 and rK39. Sera from parasitologically confirmed symptomatic VL patients (n = 90) and healthy endemic controls (n = 90) were submitted to ELISA with rK18 and rKR95. Sensitivity (95% CI) was, respectively, 83.3% (74.2­89.7) and 95.6% (88.8­98.6), and specificity (95% CI) was 93.3% (85.9­97.2) and 97.8% (91.8­99.9). For validation of ELISA with the recombinant antigens, we included samples from 122 VL patients and 83 healthy controls collected in three regions in Brazil (Northeast, Southeast, and Midwest). When comparing the results obtained with the VL patients' samples, significantly lower sensitivity was obtained by rK18-ELISA (88.5%, 95% CI: 81.5­93.2) compared with rK28-ELISA (95.9%, 95% CI: 90.5­98.5), but the sensitivity was similar comparing rKR95-ELISA (95.1%, 95% CI: 89.5­98.0), rK28-ELISA (95.9%, 95% CI: 90.5­98.5), and rK39-ELISA (94.3%, 95% CI: 88.4­97.4). Analyzing the specificity, it was lowest with rK18-ELISA (62.7%, 95% CI: 51.9­72.3) with 83 healthy control samples. Conversely, higher and similar specificity was obtained by rKR95-ELISA (96.4%, 95% CI: 89.5­99.2), rK28-ELISA (95.2%, 95% CI: 87.9­98.5), and rK39-ELISA (95.2%, 95% CI: 87.9­98.5). There was no difference in sensitivity and specificity across localities. Crossreactivity assessment, performed with sera of patients diagnosed with inflammatory disorders and other infectious diseases, was 34.2% with rK18-ELISA and 3.1% with rKR95- ELISA. Based on these data, we suggest using recombinant antigen KR95 in serological assays for VL diagnosis.

Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.

Ehrlichia canis detection in dogs from Várzea Grande: a comparative analysis of blood and bone marrow samples / Detecção de Ehrlichia canis em cães domiciliados em Várzea Grande: análise comparativa entre amostras de sangue e medula óssea

ABSTRACT: The objective of this study was to compare the DNA detection of Ehrlichia canis in blood and bone marrow to determine the prevalence of the agent in Várzea Grande, Mato Grosso. Blood samples and bone marrow from 80 dogs of both sexes, different breeds and age, were collected and processed for a cross-sectional study performed using nested PCR. Of the 80 dogs, 61 (76.3%) had E. canis DNA in one of the samples. The buffy coat was positive in 42 dogs (52.5%) and the bone marrow was positive in 33 (41.3%). There was no significant association between the positive biological samples of either the buffy coat or bone marrow and the presence or absence of clinical signs (P=0.49). No risk factor was associated with infection in the studied area. The bone marrow samples were efficient for the molecular diagnosis of canine ehrlichiosis, particularly when there was a negative blood sample, although infection was present.

RESUMO: Este trabalho teve por objetivo comparar a detecção de DNA de Ehrlichia canis em amostras de sangue e medula óssea, além de determinar a ocorrência do agente em Várzea Grande, Mato Grosso. Amostras de sangue e medula óssea de 80 cães, de ambos os sexos, diferentes raças e idade, foram coletados em estudo seccional e processados para realização de nested PCR. Dos 80 cães, 61 (76,3%) apresentaram DNA de E. canis em uma das amostras pesquisadas. A capa leucocitária foi positiva em 42 (52,5%) e a medula óssea em 33 (41,3%). Não foi observada associação significativa com a positividade das amostras biológicas, sangue ou medula óssea, e a presença ou ausência de sinais clínicos (P=0,49). Nenhum fator de risco foi associado à infecção na área pesquisada. A amostra de medula óssea mostrou-se bom sítio para o diagnóstico molecular da erliquiose canina, principalmente quando da infecção com negatividade da amostra sanguínea.

Pythium insidiosum colitis in a dog: treatment and clinical outcome / Colite por Pythium insidiosum em um cão: tratamento e evolução clínica

ABSTRACT: The aim of this report is to describe the clinical, pathological and imaging findings and treatment of colitis caused by Pythium insidiosum in a canine presenting haematochezia and progressive weight loss. Through imaging, a thickening of the transverse and descending colon was observed. Histopathological analysis of the large intestine fragment revealed the presence of hyphae, confirmed by immunohistochemistry and PCR as P. insidiosum. Antifungal treatment with itraconazole implemented after partial surgical resection, resulted in control of the disease.

RESUMO: O objetivo deste relato é descrever os achados clínico-patológicos, de imagem e o tratamento de colite por Pythium insidiosum em canino apresentando hematoquezia e emagrecimento progressivo. Nos exames de imagem, foram observados espessamento do cólon transverso e descendente. Análise histopatológica de fragmento do intestino grosso revelou a presença de hifas, confirmado pela imuno-histoquímica e PCR como P. insidiosum. Terapia antifúngica com itraconazol foi instituída pós-ressecção cirúrgica parcial, obtendo-se controle da doença.