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.

Serum IgA Antibodies Specific to M. leprae Antigens as Biomarkers for Leprosy Detection and Household Contact Tracking.

Leprosy remains endemic in several developing countries, such as India and Brazil, in part due to delayed diagnosis that facilitates ongoing transmission. Although immunoglobulins against several Mycobacterium leprae antigens have been indicated for the early diagnosis, and IgA participates in the early stages of leprosy and in subclinical infection, relatively little research has examined anti-M. leprae IgA responses. Here, we investigated serum IgA reactivity against NDO-HSA, LID-1 and NDO-LID, in paucibacillary (PB) and multibacillary (MB) patients and their household contacts, using enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy of each ELISA was evaluated by receiver operating characteristic (ROC) curve analysis. Our data reveal elevated IgA serum levels against the three M. leprae specific antigens in MB patients, whereas IgA reactivity in PB patients was increased only to NDO-HSA. Further, MB and PB household contacts displayed higher IgA reactivity to NDO-HSA than non-endemic controls. Our data suggest measurement of serum IgA against NDO-HSA as an additional tool in the diagnosis and classification of the disease, with potential utility for household contact follow-up.

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.

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.