Single (unpaired) samples used in IgG subclass comparisons, and clinical status of the Indian and Sudanese patient groups.

<p>EHC = endemic healthy control; PKDL = post kala-azar dermal leishmaniasis.</p>a<p>treated, not in recent past, but time of treatment unknown.</p><p>Single (unpaired) samples used in IgG subclass comparisons, and clinical status of the Indian and Sudanese patient groups.</p

RDT IgG1 prototypes show the ability to distinguish relapsed VL from cured VL.

<p>C = migration control line; T = test line. Black dots indicate places where samples should be deposited: 2 µl serum in front of upper dot for prototype 1 or single dot in prototype 2, and 2 µl of buffer in front of lower dot in prototype 1.</p

Specific IgG1 ELISA levels were high in active and relapsed VL but negative or substantially decreased in cured VL using unpaired serum samples.

<p>[A] Indian VL pilot study (Trial 1). [B] Indian VL expanded study (Trial 2). [C] Sudanese VL. Mean and 95% CI are shown for each data set (solid black lines); note the different Y axis scales. In each study set, the means plus three standard deviations obtained using DAT-seronegative endemic healthy control (EHC) samples was used to calculate the cut-off value (dotted line) and p values of<0.05 were considered significant.</p

Summary of results from IgG1 rapid diagnostic tests (RDT) prototypes.

a<p>Therapy: sodium antimony gluconate n = 8; miltefosine n = 10; amphotericin B n = 3; combination therapy n = 2.</p>b<p>22 of these samples were also used with prototype 1.</p>c<p>these samples were also negative with prototype 1.</p>d<p>these 5 samples were also used with prototype 1.</p>e<p>malaria n = 3; hepatitis n = 1; TB n = 2; dengue n = 1.</p><p>Summary of results from IgG1 rapid diagnostic tests (RDT) prototypes.</p

IgG subclass serology in VL and PKDL: Published studies.

<p>IgG subclass serology in VL and PKDL: Published studies.</p

IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test.

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