Development of an Improved Whole Blood Assay for Diagnosis of Latent and Active Tuberculosis Cases

Background: Latent TB infection (LTBI) is an infection where the presence of disease causing organism M. tuberculosis is there without any sign and symptoms of the disease hence mostly remains undiagnosed, though Tuberculin skin test (TST) and Interferon Gamma Release Assay (IGRA) were used to diagnose the LTBI. They have their limitations, TST gives major crossreactivity with BCG vaccine and gives inaccurate results in individuals who have taken BCG and IGRA are very costly and variable sensitivity is repeated in various populations hence the modifications are needed in the IGRA for proper diagnosis of LTBI. Objectives: In the proposed study we aimed to develop an improved whole blood assay towards a diagnosis of latent and active TB infection as an alternative to the Quantiferon QFT assay Methodology: Synthetic antigenic peptides against latency specific antigens will be designed and synthesized. Designed peptides will be screened for LTBI specific cytokine by in-vitro experiments. Development & production of Whole assay using selected peptides evaluation of developed assay through ELISA in clinical samples. Study Protocol Nashine et al.; JPRI, 33(38B): 197-209, 2021; Article no.JPRI.71198 198 Expected Results: Latent specific peptides will be identified and peptide-based whole blood assay for detection and diagnosis of tuberculosis will be developed as an indigenous alternative for the existing QFT assay. Conclusion: An improved whole blood assay will be developed for screening of LTBI in the Indian population.


Latent TB Infection (LTBI) is an infection where the presence of disease-causing organism
Mycobacterium tuberculosis is there without any sign and symptoms of the disease [1,2]. Tuberculin skin test (TST) is a standard diagnostic test for LTBI [3]. In this test, an intradermal injection of purified protein derivative (PPD) is given to the patient & hypersensitivity response in the form of cutaneous induration was measured at the site of injection at 48-72 h. However, it was reported to give a false positive result among BCG vaccinated individuals [4][5][6].
Similarly, Interferon-Gamma Release Assay (IGRAs) is an advanced immunological tube assay, which measures the Interferon-Gamma level in the blood samples of the patient in response to challenge with tuberculosis antigens [7][8][9]. In earlier studies the prevalence of LTBI observed according to QFT-G is of around 48% in India [10,11]. However variable sensitivity of IGRA is reported for diagnosis of LTBI [12,13] so, there is also need of some improved tests for diagnosis of TB infection [14,15].
The main objective of the present study is to develop an improved blood assay for the diagnosis of LTBI infection. In brief, first will design and synthesis the synthetic antigenic peptides against latency-specific antigens. Designed peptides will be screened by In-vitro experiments after which development & evaluation of whole blood assay will be done using selected peptides (Schematic plan is given in Fig. 1).

METHODOLOGY
Study site: Samples for the study will be collected from OPD patient enrolled at CIIMS hospital Nagpur, Wards of CIIMS hospital Nagpur, and through camps conducted by CIIMS at the periurban areas of Nagpur as well as Melghat Amravati with help of the satellite center of CIIMS, i.e. Dr. G.M Taori Tribal Health research center Melghat, Amravati, (M.S) India.
Thus the Place of the study will include Nagpur, & Melghat.
Study Participant: All participants will be recruited using predefined inclusion-exclusion criteria. There will be three major groups of participants for sample collection as follows:  Active TB subjects (n=62)  House hold contacts (latent cases) (n=89)  Healthy controls (n=96) Samples: Blood samples (6-8ml) in sterile EDTA tubes and QFT tubes (1 to 2 ml each tube)

Expected Outcomes
IGRA done in the subjects will give the idea about the subjects to be included in the latent category based on the positivity as described in inclusion-exclusion criteria and history.

TUBERCULIN SKIN TESTS (TST)
Tuberculin Skin Test (TST) is been developed and provided by MDH (Minnesota Department of Health). TST is a test used for the detection of M .tuberculosis. The test will be performed and used for the identification of sample groups i.e. Positive, Latent, and control groups [17] ( Table 2).

DESIGNING ANTIGENIC PEPTIDES AGAINST LATENCY SPECIFIC ANTIGENS OF M. tuberculosis
For designing of antigenic peptides against latency specific antigens of M. Tuberculosis.

Procedure
1. The sequence of Latency specific protein (Rv1978, Rv2623, Rv2029, Rv3133, Rv2624c, Rv2626c, and Rv2628, etc) will be extracted from proteomic data repository for pathogenic mycobacteria i.e Mycobrowser (Mycobacterial browser) (https://mycobrowser.epfl.ch/) 2. The antigenic peptide in each targeted protein molecule will be predicted using the immune epitope database (IEDB) website  Latent TB specific protein sequence will be taken from mycobrowser will be then entered to predict T cell epitopes for MHC class I and II binding.  The IEDB recommended prediction method (Default method) will be used for the prediction of the antigen.  The results will be obtained from the iedb.org via email. 3. The results of several peptides with information like rank, score, start, and endpoint along with the length of peptides will be obtained. 4. As per IEDB recommendation, the peptides with less than 1 rank will be filtered for the further analysis 5. Repeated/Duplicate peptides will be removed from the obtained results sheet 6. These peptides will be then selected for checking the MHC Class I and II immunogenicity scores by the use of the IEDB website in the epitope analysis resource section 7. The top 25% peptides with the highest immunogenicity score will be then selected from each protein set 8. By applying all the above parameters peptides will be selected for the blast analysis. 9. The peptide, which will be found nonspecific in Blast analysis, will be excluded and the remaining peptide will be sent for synthesis.

SYNTHESIS OF PEPTIDE
Based on Blast result the most potent and specific peptides will be selected and synthesize, through custom services with suitable company.  The PBMC is consists of leukocytes which are isolated from whole blood and contain lymphocytes, monocytes, and a small percentage of some other immune cells, such as dendritic cells. PBMCs can be stimulated or treated with or drugs in-vitro [18][19]. Peripheral blood lymphocytes from heparinized blood will be isolated by gradient centrifugation using HISTOPAQUE -1077 ( Fig. 4)

CELL VIABILITY AND CELL COUNT ASSAY
Trypan blue staining is used to check cell membrane integrity and cell viability. The isolated and washed lymphocytes are checked for viability and cell count using a heamocytometer.

Principle
The viable and living cells do not dye in Trypan blue staining as they expel the dye intruding the cell as metabolically active cells while non-viable and dead cells fail to do so and stain blue in Trypan `Blue staining.

In vitro SCREENING OF DESIGNED PEPTIDES
PBMC will be incubated in the presence and absence of different concentrations of peptide and its cocktails.

Note
 Cell concentration for the experiment will be optimized  LPS concentration will be used as reported in the literature  The concentration of each peptide will be optimized

Principle
In XTT assay, Mitochondrial enzymes of the live cells reduce the tetrazolium salt into orange colored compounds of formazan dye, which directly proportional to the live cells. The dye  Collect the supernatant & pallet and store c until further use (or proceed with (Table 5) Cell concentration for the experiment will concentration will be used as reported The concentration of each peptide will be

XTT ASSAY)
In XTT assay, Mitochondrial enzymes of the live salt into orangecolored compounds of formazan dye, which directly proportional to the live cells. The dye formed is water-soluble and can be easily read at 450nm. The assay is mainly used for assessment of the cell viability and cytotoxicity, in comparison to the untreated control set (Table  6).

Reagents
XTT Reagent: 10 mg XTT is dissolved in 10ml warm PBS. Aliquots are prepared in vials and are stored at -20ºC.
[XTT-2,3bis (2-methoxy-4 nitro 5tetrazolium-5 carboxanilide sodium sal soluble and can be easily read at 450nm. The assay is mainly used for assessment of the cell viability and cytotoxicity, on to the untreated control set (Table   10 mg

Possible solution
To avoid contamination UV treatment should be given to the equipment in the Biosafety cabinet for at least 20min  Table 6. Trouble Shooting of cell viability assay (XTT assay) Step Possible problem Possible reason Possible solution 3 No proper results obtained XTT reagent used alone without activation reagent Prepare XTT reagent along with activation reaction solution and repeat assay 6 Optical density too high Cell numbers per well are too high Reduce number of cells plated per well

Calculation
ROC curve analysis will be performed over the cytokine value observed in a known set of Latent, Active, and Control blood samples, using statistical software for calculation of cut off value.

EVALUATION OF IMPROVED WHOLE BLOOD ASSAY
For evaluation, the developed assay will be compared with the existing, QFT assay & TST assay using the statistical tools.

STATISTICAL ANALYSIS
All the analysis will be performed using standard statistical software. The receiver operating curve (ROC) will be used for determining the cut-off values for individual cytokines. Subjects who will have cytokine and chemokine levels above the cut-off point will be considered as positive.

RESULTS
IGRA done in the subjects will give the idea about the subjects to be included in the latent category based on the positivity as described in inclusion-exclusion criteria and history. Latent specific peptides will be identified and synthesized for further evaluation through In-vitro protocols and will be further used for the development of whole blood assay. From the collected samples the PBMC will be isolated which further will be useful for in vitro screening of designed peptides. Identification of cytokine panel for diagnosis of the LTBI will take place and these identified cytokines will be used in the development of improved whole blood assay. By doing cell count the accurate number of cells (viable and non-viable) present in 1ml of pellet will be obtained which will be useful in In-vitro screening of the designed peptides. Evaluation of multi-cytokine in response to the disease-specific antigen will help us in understanding the role of other cytokine involved in latency and active infection. Also, our peptide-based approach may reduce the cost of developed Whole Blood Assay.

DISCUSSION
One of the study mentioned that there are significant differences in IFN-γ responses for DosR antigens (Rv1735c, Rv2006, Rv2625c, Rv1996, Rv2032, Rv2629, Rv3126c, Rv0081, Rv2631, Rv3130c, Rv2624c, Rv2007c, Rv2028c, and Rv3134) in healthy household contacts compared with patients with active TB, as reported by a study in whole blood that included a wide range of stage-specific antigens to assess IFN-γ response in a long-incubation assay [20].
In the similar way we will identify major DosR antigens based on literature and will extract their peptides which will be further filtered by the use of online software's such as www.idbi.org and other online software's.

CONCLUSION
Thus in our study further evaluation of the filtered potential peptides will be done and these peptides will be analyzed by In-Vitro studies using the peripheral blood mononuclear cells (PBMC) in the animal cell culture laboratory to see the cytokine response of these peptides. The identified potential immunogenic peptide target will be useful for studying the patho-physiology, of latent tuberculosis & development of alternative diagnostic protocol, for Latent TB infection.

LIMITATIONS
IGRA's are developed and evaluated in the western population hence are less sensitive and specific for the Indian population and there is a need for its modification and also IGRA is a costly affair.

CONSENT
As per international standard or university standard, patient's written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
The study was approved by the Institutional Ethical Committees of both, Central India Institute of Medical Science (CIIMS) Nagpur and Datta Meghe Institute of Medical Sciences (DMIMS), Sawangi, Wardha.