Browsing by Author "Michael A. Miles"

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Detection of Immunoglobulin G1 against rK39 Improves Monitoring of Treatment Outcomes in Visceral Leishmaniasis
(Oxford University Press, 2019) Guy Mollett; Bruno C. Bremer Hinckel; Tapan Bhattacharyya; Tegwen Marlais; Om Prakash Singh; Pascal Mertens; Andrew K. Falconar; Sayda El-Safi; Shyam Sundar; Michael A. Miles
Background: Visceral leishmaniasis (VL), caused by the Leishmania donovani complex, is a fatal, neglected tropical disease that is targeted for elimination in India, Nepal, and Bangladesh. Improved diagnostic tests are required for early case detection and for monitoring the outcomes of treatments. Previous investigations using Leishmania lysate antigen demonstrated that the immunoglobulin (Ig) G1 response is a potential indicator of a patient's clinical status after chemotherapy. Methods: IgG1 or IgG enzyme-linked immunosorbent assays (ELISAs) with rK39 or lysate antigens and novel IgG1 rK39 rapid diagnostic tests (RDTs) were assessed with Indian VL serum samples from the following clinical groups: paired pre-and postchemotherapy (deemed cured); relapsed; other infectious diseases; and endemic, healthy controls. Results: With paired pre-and post-treatment samples (n = 37 pairs), ELISAs with rK39-and IgG1-specific conjugates gave a far more discriminative decrease in post-treatment antibody responses when compared to IgG (P <. 0001). Novel IgG1 rK39 RDTs provided strong evidence for decreased IgG1 responses in patients who had successful treatment (P <. 0001). Furthermore, both IgG1 rK39 RDTs (n = 38) and ELISAs showed a highly significant difference in test outcomes between cured patients and those who relapsed (n = 23; P <. 0001). RDTs were more sensitive than corresponding ELISAs. Conclusions: We present strong evidence for the use of IgG1 in monitoring treatment outcomes in VL, and the first use of an IgG1-based RDT using the rK39 antigen for the discrimination of post-treatment cure versus relapse in VL. Such an RDT may have a significant role in monitoring patients and in targeted control and elimination of this devastating disease. © 2018 The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.
IgG1 as a Potential Biomarker of Post-chemotherapeutic Relapse in Visceral Leishmaniasis, and Adaptation to a Rapid Diagnostic Test
(Public Library of Science, 2014) Tapan Bhattacharyya; Armon Ayandeh; Andrew K. Falconar; Shyam Sundar; Sayda El-Safi; Marissa A. Gripenberg; Duncan E. Bowes; Caroline Thunissen; Om Prakash Singh; Rajiv Kumar; Osman Ahmed; Osama Eisa; Alfarazdeg Saad; Sara Silva Pereira; Marleen Boelaert; Pascal Mertens; Michael A. Miles
Background:Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care.; Methodology/Principal Findings:All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001).; Conclusions/Significance:Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse. © 2014 Bhattacharyya et al.
Isolation and characterisation of Leishmania donovani protein antigens from urine of visceral leishmaniasis patients
(Public Library of Science, 2020) Tegwen Marlais; Tapan Bhattacharyya; Callum Pearson; Bathsheba L. Gardner; Safiyyah Marhoon; Stephanie Airs; Kiera Hayes; Andrew K. Falconar; Om Prakash Singh; Steven G. Reed; Sayda El-Safi; Shyam Sundar; Michael A. Miles
Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease. © 2020 Marlais et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Refining wet lab experiments with in silico searches: A rational quest for diagnostic peptides in visceral leishmaniasis
(Public Library of Science, 2019) Bruno Cesar Bremer Hinckel; Tegwen Marlais; Stephanie Airs; Tapan Bhattacharyya; Hideo Imamura; Jean-Claude Dujardin; Sayda El-Safi; Om Prakash Singh; Shyam Sundar; Andrew Keith Falconar; Bjorn Andersson; Sergey Litvinov; Michael A. Miles; Pascal Mertens
Background The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a crude lysate antigen (CLA) as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. Methodology Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. Results Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. Conclusion We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL. © 2019 Bremer Hinckel et al.
Significantly Lower Anti-Leishmania IgG Responses in Sudanese versus Indian Visceral Leishmaniasis
(Public Library of Science, 2014) Tapan Bhattacharyya; Duncan E. Bowes; Sayda El-Safi; Shyam Sundar; Andrew K. Falconar; Om Prakash Singh; Rajiv Kumar; Osman Ahmed; Marleen Boelaert; Michael A. Miles
Background:Visceral leishmaniasis (VL), a widely distributed systemic disease caused by infection with the Leishmania donovani complex (L. donovani and L. infantum), is almost always fatal if symptomatic and untreated. A rapid point-of-care diagnostic test for anti-Leishmania antibodies, the rK39-immunochromatographic test (rK39-ICT), has high sensitivity and specificity in South Asia but is less sensitive in East Africa. One of the underlying reasons may be continent-specific molecular diversity in the rK39 antigen within the L. donovani complex. However, a second reason may be differences in specific IgG anti-Leishmania levels in patients from different geographical regions, either due to variable antigenicity or immunological response.Methodology/Principal Findings:We determined IgG titres of Indian and Sudanese VL patients against whole cell lysates of Indian and Sudanese L. donovani strains. Indian VL patients had significantly higher IgG titres against both L. donovani strains compared to Sudanese VL patients (p<0.0001). Mean reciprocal log10 50% end-point titres (1/log10t50) were i) 3.80 and 3.88 for Indian plasma and ii) 2.13 and 2.09 for Sudanese plasma against Indian and Sudanese antigen respectively (p<0.0001). Overall, the Indian VL patients therefore showed a 46.8-61.7 -fold higher mean ELISA titre than the Sudanese VL patients. The higher IgG titres occurred in children (<16 years old) and adults of either sex from India (mean 1/log10t50: 3.60-4.15) versus Sudan (mean 1/log10t50: 1.88-2.54). The greatest difference in IgG responses was between male Indian and Sudanese VL patients of ≥ 16 years old (mean 1/log10t50: 4.15 versus 1.99 = 144-fold (p<0.0001).Conclusions/Significance:Anti-Leishmania IgG responses among VL patients in Sudan were significantly lower than in India; this may be due to chronic malnutrition with Zn2+ deficiency, or variable antigenicity and capacity to generate IgG responses to Leishmania antigens. Such differential anti-Leishmania IgG levels may contribute to lower sensitivity of the rK39-ICT in East Africa. © 2014 Bhattacharyya et al.
Visceral Leishmaniasis IgG1 Rapid Monitoring of Cure vs. Relapse, and Potential for Diagnosis of Post Kala-Azar Dermal Leishmaniasis
(Frontiers Media S.A., 2018) Tegwen Marlais; Tapan Bhattacharyya; Om Prakash Singh; Pascal Mertens; Quentin Gilleman; Caroline Thunissen; Bruno C. Bremer Hinckel; Callum Pearson; Bathsheba L. Gardner; Stephanie Airs; Marianne de la Roche; Kiera Hayes; Hannah Hafezi; Andrew K. Falconar; Osama Eisa; Alfarazdeg Saad; Basudha Khanal; Narayan Raj Bhattarai; Suman Rijal; Marleen Boelaert; Sayda El-Safi; Shyam Sundar; Michael A. Miles
Background: There is a recognized need for an improved diagnostic test to assess post-chemotherapeutic treatment outcome in visceral leishmaniasis (VL) and to diagnose post kala-azar dermal leishmaniasis (PKDL). We previously demonstrated by ELISA and a prototype novel rapid diagnostic test (RDT), that high anti-Leishmania IgG1 is associated with post-treatment relapse versus cure in VL. Methodology: Here, we further evaluate this novel, low-cost RDT, named VL Sero K-SeT, and ELISA for monitoring IgG1 levels in VL patients after treatment. IgG1 levels against L. donovani lysate were determined. We applied these assays to Indian sera from cured VL at 6 months post treatment as well as to relapse and PKDL patients. Sudanese sera from pre- and post-treatment and relapse were also tested. Results: Of 104 paired Indian sera taken before and after treatment for VL, when deemed clinically cured, 81 (77.9%) were positive by VL Sero K-SeT before treatment; by 6 months, 68 of these 81 (84.0%) had a negative or reduced RDT test line intensity. ELISAs differed in positivity rate between pre- and post-treatment (p = 0.0162). Twenty eight of 33 (84.8%) Indian samples taken at diagnosis of relapse were RDT positive. A comparison of Indian VL Sero K-SeT data from patients deemed cured and relapsed confirmed that there was a significant difference (p < 0.0001) in positivity rate for the two groups using this RDT. Ten of 17 (58.8%) Sudanese sera went from positive to negative or decreased VL Sero K-SeT at the end of 11–30 days of treatment. Forty nine of 63 (77.8%) PKDL samples from India were positive by VL Sero K-SeT. Conclusion: We have further shown the relevance of IgG1 in determining clinical status in VL patients. A positive VL Sero K-SeT may also be helpful in supporting diagnosis of PKDL. With further refinement, such as the use of specific antigens, the VL Sero K-SeT and/or IgG1 ELISA may be adjuncts to current VL control programmes. © Copyright © 2018 Marlais, Bhattacharyya, Singh, Mertens, Gilleman, Thunissen, Hinckel, Pearson, Gardner, Airs, de la Roche, Hayes, Hafezi, Falconar, Eisa, Saad, Khanal, Bhattarai, Rijal, Boelaert, El-Safi, Sundar and Miles.