TITLE:
A Novel Urine Method for the Diagnosis of Active Tuberculosis by Immunoassay for the Detection of ESAT-6 Using Hydrogel Nanoparticles in HIV Patients
AUTHORS:
Luz Huaroto, Raquel Mugruza, Marinelly Roca, Ricardo Antiparra, César Ticona, Patricia Sheen, Eduardo Ticona, Robert H. Gillman, Alessandra Luchini, Lance Liotta
KEYWORDS:
Tuberculosis, HIV+, Hydrogel Nanoparticles and ESAT-6
JOURNAL NAME:
Journal of Tuberculosis Research,
Vol.9 No.2,
April
25,
2021
ABSTRACT: Background: In patients with HIV, conventional tests are of low sensitivity; therefore, a
new diagnostic test with hydrogel nanoparticles with reactive blue dye is
proposed, which allows the capture, conservation and concentration of ESAT-6 in
urine. NIPAs are copolymers that capture low molecular weight proteins and
protect against enzymatic degradation. Using an immunoassay, it is possible to
detect and quantify ESAT-6 present in urine as a possible marker of active TB. Design/Methods: Study in Lima, Peru, HIV+ participants, ≥18 years with and without tuberculosis
(TB). Smear microscopy, culture in solid medium and urine immunoassay were
performed. The reference was the diagnosis of TB by radiological or clinical
microbiological criteria (indication for TB treatment). There were 2
preanalytical processes: untreated and treated urine (centrifuged, heated),
then incubation with NIPAm. After washing, elution, sonication, heat and
centrifugation, the final eluate was obtained. This was placed on
nitrocellulose membranes, which by means of fixation and incubation processes
with anti-ESAT-6 and anti-IgG antibodies, revelation with C-DiGit® Blot
Scanner and FluorChem R FR0001. Calibration curves were included in the
membranes, density was measured using Image J software. ROC curves, sensitivity
and specificity were obtained. Results: The result by
groups was HIV+ patient: ROC: 0.75, cut-off
point ≥24.06 ng/ml, sensitivity 76.32%, specificity 68.89%, patients ≤200 cells
CD4 mm3/ml, ROC: 0.78, cutoff point ≥26.20 ng/ml, sensitivity
75.86%, specificity 71.88%, patients >200
CD4 mm3 cells/ml, ROC: 0.73,
cutoff point ≥24.6 CD4 mm3/ml, sensitivity 73.68%,
specificity 73.68%. Conclusions: The ESAT-6 detection
assay using NIPAm was effective, with higher rates in patients with ≤200 CD4
cells/mm3, the test being more sensitive than smear and
culture, but less specific.