A Novel Urine Method for the Diagnosis of Active Tuberculosis by Immunoassay for the Detection of ESAT-6 Using Hydrogel Nanoparticles in HIV Patients

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 mm/ml, ROC: 0.78, cutoff point ≥26.20 ng/ml, sensitivity 75.86%, specificity 71.88%, paHow to cite this paper: Huaroto, L., Mugruza, R., Roca, M., Antiparra, R., Ticona, C., Sheen, P., Ticona, E., Gillman, R.H., Luchini, A. and Liotta, L. (2021) A Novel Urine Method for the Diagnosis of Active Tuberculosis by Immunoassay for the Detection of ESAT-6 Using Hydrogel Nanoparticles in HIV Patients. Journal of Tuberculosis Research, 9, 73-84. https://doi.org/10.4236/jtr.2021.92006 Received: January 13, 2021 Accepted: April 22, 2021 Published: April 25, 2021 Copyright © 2021 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Currently, tuberculosis (TB) and human immunodeficiency virus (HIV) infection are two of the leading causes of infectious mortality worldwide [1]. Approximately 25% of the world's population are infected with TB and at possible risk of active disease. In 2018, the World Health Organization (WHO) reported 10 million new TB cases, of which 8.6% also had HIV infection [2], approximately 251,000 deaths. In Peru, it is the main cause of death in the HIV patients TB [3] [4] [5].
Patients HIV positive are highly susceptible to tuberculosis infection, due to their compromised immune system, and therefore low immune response, which does not form caverns, therefore, his patients had a decreased proliferation of bacilli, that difficult the conventional test like direct bacilli observation, culture and nucleic acid amplification [6] [7] [8]. This is the main reason for late diagnosis and treatment initiation, increasing the probability of M. Tuberculosis spread to other organs.
Currently, in low and middle countries the diagnosis of TB in HIV population is based on smear ZN staining, a test with low sensitivity 28%, cultures (Ogawa, MODS and Bactec) have sensitivity greater than 68%, however takes a long incubation period and need high biosecurity measures [9] [10] [11] [12]. Molecular tests such as Xpert MTB/RIF 79%, genotype 30% are very effective, but high cost, also, are aimed more to determine drug sensitivity than diagnosis. Therefore, its implementation and use in low complexity laboratories is not very feasible [13] [14]. Radiology diagnosis is not very effective in HIV patients because they don't present the conventional radiologic patrons.
Several studies are searching active TB markers in other samples than sputum, focus on M. tuberculosis antigens detection in urine, due to its easy sample collection, less risk of aerosols for laboratory personnel and lower cost per application and use in a low complexity laboratory. However, the enzymatic degradation of antigens in urine doesn't allow the antigen detection [15].
Other tests have been developed, such as the lipoarabinomannan detection (LAM) in urine by immunochromatography, which is applied in patients with HIV infection with <200 CD4 mm 3 cells with a sensitivity of 4.0% and up to 66.7% in <50 CD4 mm 3 cells [12]. Due to this, its use is diminished and mainly Due to the need to develop new simple, fast and easy-to-implement diagnostic tests, with greater sensitivity and specificity a new method is with the use of hydrogel nanoparticles for the determination of ESAT antigen-6. Profiling as one of the easiest to apply due to its ease in obtaining the sample and the possible greater sensitivity than other tests.
For these reasons, the main objective of this study is to determine a diagnostic method for active TB in patients with HIV co-infection, through the detection of ESAT-6.  In some membranes with an unclear and therefore invalid calibration curve, the equation of a valid standard curve of a membrane performed on the same day (same eluates of the calibration curve) was used.

Ethical Considerations
The protocol was approved by the

Conclusions
For the study groups, AUC value was greater >70, sensitivity >60% and specificity >60% were found. The group with the best rates was for the diagnosis of pulmonary tuberculosis in patients with HIV infection.
Urine samples are easy and quicker to obtain, is only necessary refrigeration Journal of Tuberculosis Research as soon as possible. Is plausible method, with lower risk of handling potentially infectious samples. Likewise, by detection of a TB marker, decreases high exposure to health personnel by obtaining and managing samples like bronchial aspirates, biopsies, or induced in HIV patients.
In the study, two preanalytical procedures were analyzed for treated and untreated urine both calibration curves were validated. However, there was no significant difference between the calibration curves of both methods. The untreated urine protocol requires fewer preanalytical conditions and is more feasible for field application. However, in recent years, health centers have more equipment, making it possible to use a thermoblock and a centrifuge, where samples can be processed into pellets for transport in the laboratory. Although the nanoparticles are stable at room temperature and could be used in the field, the protocol for treated urine and its use in the laboratory allows us to include the initial solution (urine) and the supernatant in the immunoassay, which serves as an additional internal control of the test.
In the study, the development of the immunoassay was carried out in two equipment, the method was validated in both FluorChem R FR0001 and C Digit Blot Scanner equipment, however, a difference between the thresholds was detected. For the application of the test, the method must be validated using the available chemiluminescence reader.
The immunoassay procedure takes up to 12 hours, despite the duration of the procedure, it is considerably less than an incubation time of a culture and at a lower cost of a molecular test. More experiments are necessary to decrease the time in the procedures in order for the method to be more adaptable to the clinical field.
The sensitivity and specificity values of the NIPA ESAT-6 urine test are greater than 70%, more sensitive, less specific than smear microscopy (sensitivity 28%, specificity 92%) and less sensitive or specific than a culture (sensitivity 80% -90%, specificity 98%) in the HIV population, all with an AUC value >73%, classifying it as a good test.
Several studies have tested the detection of new biomarkers in both sputum and urine, thus, in 2017, a study testing a cocktail immunoassay of antigens such as ESAT-6, CFP10 and MPT64 in urine, the sensitivities were low 68%, 2%, 22% and 31.6%, respectively [26].
In Indonesia, they carried out a qualitative detection study of antigens by immunochromatography such as ESAT-6, CFP-10 and MPT64, a 78% specificity and 68.8% sensitivity were determined, determining that the markers should more study [2].
In Geneva, performed a new electrochemiluminescence method for the detection of ESAT-6 in urine and serum, the method presented a minimum detection threshold as pg/ml. In urine, the sensitivity of LAM and ESAT-6 were 93% and 65%, respectively. For ESAT-6, 55% and 46% were presented, respectively [27].
Likewise, there is the Silva LAMP, for the detection of LAM for the diagnosis of extrapulmonary tuberculosis, which determined a sensitivity of 60% for Pul- Due to this, in 2013, an immunoassay with gold nanoparticles, an immunoassay (ELISA) was carried out for the detection of ESAT-6 where promising results were presented, this type of nanoparticles presented a concentration factor of 7.5×. However, the hydrogel nanoparticles showed greater stability and a concentration factor of up to 50× [21].
The study method determined to be applicable for the diagnosis of TB in all locations, however, better rates were determined for the diagnosis of pulmonary TB, this possibly due to misclassification bias in patients with extrapulmonary TB due to the low sensitivity of the tests for the TB diagnosis and clinical assessment for initiation of treatment. To determine the sensitivity and specificity of the extrapulmonary TB test, confirmatory studies by molecular or microbiological biology with more sensitive methods are necessary, as well as a larger sample size. However, the results of the study allow us to say that this test is possibly even more sensitive than current conventional tests. There are molecular tests that have determined a sensitivity level greater than 40% in the non-HIV population. However, in a population with coinfection, it may have a lower level.
As well as the difficulty of applying mPCR tests [25].
The present study of nanoparticles shows promising results of sensitivity, the test can be used as a TB screening that must be interpreted together with a clinical and epidemiological evaluation, thus reducing the delay in the start of treatment. However, larger studies of latent TB should be performed in the same population to eliminate possible classification bias.

Funding
Study is financed by the Basic and Applied Sciences Project Contest of FONDECYT-Lima, Peru (Contract 120-2016).

Conflicts of Interest
The authors declare no conflicts of interest.