Immuno-PCR, a new technique for the serodiagnosis of tuberculosis

Accepted Manuscript Immuno-PCR, a new technique for the serodiagnosis of tuberculosis

Promod K. Mehta, Bhawna Dahiya, Suman Sharma, Netrapal Singh, Renu Dharra, Zoozeal Thakur, Neeru Mehta, Krishna B. Gupta, Mahesh C. Gupta, Dhruva Chaudhary PII: DOI: Reference:

S0167-7012(17)30119-7 doi: 10.1016/j.mimet.2017.05.009 MIMET 5165

To appear in:

Journal of Microbiological Methods

Received date: Revised date: Accepted date:

14 March 2017 16 May 2017 16 May 2017

Please cite this article as: Promod K. Mehta, Bhawna Dahiya, Suman Sharma, Netrapal Singh, Renu Dharra, Zoozeal Thakur, Neeru Mehta, Krishna B. Gupta, Mahesh C. Gupta, Dhruva Chaudhary , Immuno-PCR, a new technique for the serodiagnosis of tuberculosis, Journal of Microbiological Methods (2017), doi: 10.1016/j.mimet.2017.05.009

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REVISED Immuno-PCR, a new technique for the serodiagnosis of tuberculosis Promod K. Mehta1*, Bhawna Dahiya1, Suman Sharma1, Netrapal Singh1, Renu Dharra1, Zoozeal

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Thakur1, Neeru Mehta2, Krishna B. Gupta3, Mahesh C. Gupta4 and Dhruva Chaudhary5

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1. Centre for Biotechnology, Maharshi Dayanand University (MDU), Rohtak-124001 (Haryana), India. 2. Department of Medical Electronics, Ambedkar Institute of Technology, Shakarpur,

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Delhi-110052, India. 3. Department of TB and Respiratory Medicine, Postgraduate Institute of Medical Sciences (PGIMS), Rohtak-124001. 4. Department of Pharmacology, PGIMS, Rohtak-

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124001, India. 5. Pulmonary and Critical Care Medicine, PGIMS, Rohtak-124001, India.

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* Corresponding Author: Promod K. Mehta, Centre for Biotechnology, Maharshi Dayanand

E-mail: [email protected]

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Tel # 91-9896504193

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University, Rohtak-124001 (Haryana), India.

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Fax # 91-1262-274640

Key words: Mycobacterium tuberculosis, Tuberculosis, Immuno-PCR, Serodiagnosis, Antigen

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Detection, Antibody Detection

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Running Title: Serodiagnosis of TB by immuno-PCR

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ACCEPTED MANUSCRIPT Abstract

Rapid and accurate diagnosis of tuberculosis (TB) is essential to control the disease. The conventional microbiological tests have limitations and there is an urgent need to devise a simple, rapid and reliable point-of-care (POC) test. The failure of TB diagnostic tests based on antibody detection due to inconsistent and imprecise results has stimulated renewed interest in

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the development of rapid antigen detection methods. However, the world health organization

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(WHO) has emphasized to continue research for designing new antibody-based detection tests

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with improved accuracy. Immuno-polymerase chain reaction (I-PCR) combines the simplicity and versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential

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amplification capacity and sensitivity of PCR thus leading to several-fold increase in sensitivity in comparison to analogous ELISA. In this review, we have described the serodiagnostic

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potential of I-PCR assays for an early diagnosis of TB based on the detection of potential

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mycobacterial antigens and circulating antibodies in body fluids of TB patients.

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ACCEPTED MANUSCRIPT Contents 1. Introduction 1.1.

Evolution of immuno-PCR (I-PCR)

1.2.

Different formats of I-PCR

2. Antigen detection Antigen detection by I-PCR

2.2.

Antigen detection by RT-I-PCR

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2.1.

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3. Antibody detection

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4. Merits 5. Demerits

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6. Conclusion

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Abbreviations: Ag85, antigen 85; BCG, M. bovis bacillus Calmette-Guérin; CRS, composite reference standard; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; EPTB,

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extrapulmonary TB; GNP, gold nanoparticles; HIV, human immunodeficiency virus; IGRA, interferon-gamma release assay; IL-PCR, Immunoliposome-PCR; I-PCR, immuno-polymerase

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chain reaction; LAM, lipoarabinimannan; NPA-I-PCR, nanoparticle amplified I-PCR;

NTM,

nontuerculous mycobacteria; PD-I-PCR, phage display-mediated I-PCR; PEG, polyethylene

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glycol; PTB, pulmonary tuberculosis; RD, regions of differences; RT-I-PCR, real-time I-PCR; SATA, N-succinimidyl-S-actyl-thioacetate;

SMCC, succinimidyl 4-[N-maleimidomethyl]-

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cyclohexane-1-carboxylate; TB, tuberculosis; WHO, World health organization.

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ACCEPTED MANUSCRIPT 1. Introduction

Tuberculosis (TB) is a major public health problem worldwide ranking above the human immunodeficiency virus (HIV) infection as the leading cause of deaths from infectious diseases. In 2015, approximately 10.4 million new TB cases were reported including 1.2 million HIVpositive individuals (WHO, 2016). In developing countries like India, diagnosis of pulmonary

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tuberculosis (PTB) is mainly dependent on sputum smear examination, which is simple but the

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sensitivity is only 50–60% (Haldar et al., 2011; Siddiqi et al., 2003). Though culture

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identification is considered as the gold standard, it has a turnaround time of 4–8 weeks and requires skillful technicians. During the last two decades, an unprecedented interest has arisen in

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designing new TB diagnostic tools including nucleic acid amplification tests such as polymerase chain reaction (PCR) and interferon-gamma release assays (IGRAs, Cattamanchi et al., 2011;

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Mehta et al., 2012b), however, these tests have limitations. PCR tests targeting IS6110, mpb64 (Rv1980c), pstS1 (Rv0934), devR (Rv3133c), etc. are widely used especially for the diagnosis of

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paucibacillary extrapulmonary TB (EPTB) specimens (Haldar et al., 2011; Mehta et al., 2012b; Raj et al., 2016), but lead to variable sensitivities and cannot detect non-nucleic acid molecules

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that are abundantly present in TB samples and remain unexplored. Recently, GeneXpert assay, a heminested real-time PCR targeting rpoB (Rv0664) encoding RNA β polymerase subunit has

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been a major breakthrough in TB diagnostics, which identifies Mycobacterium tuberculosis and rifampicin resistance simultaneously; however, its wide implementation and scale-up is restricted

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in resource-poor settings primarily due to its high cost and the need for a continuous and stable power supply to operate the GeneXpert instrument (Denkinger et al., 2014; Heidebrecht et al.,

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2016). Similarly, IGRAs cannot distinguish between active disease and latent infection and are considered expensive for resource-poor settings.

1.1 Evolution of immuno-PCR (I-PCR)

Immuno-PCR (I-PCR) is basically similar to ELISA, but instead of using an enzyme-conjugated antibody, the antibody is labeled with DNA fragment through streptavidin-biotin conjugates or by a covalent binding, and which is amplified by PCR (Fig. 1a, b). Though ELISA is the most commonly used method for the detection of mycobacterial antigens and antibodies, it fails when 4

ACCEPTED MANUSCRIPT there is low concentration of target antigens/antibodies (Mehta et al., 2014; Spengler et al., 2015). Originally discovered by Sano et al. (1992), I-PCR could detect as few as 580 bovine serum albumin molecules, wherein the detection antibody was coupled to the biotinylated DNA molecule through streptavidin-protein A chimeric molecule. However, the limited availability of streptavidin–protein A fusion protein and the widely varied affinities of protein A with antibodies of various classes and subclasses from different species restricted its immediate use.

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To overcome this problem, Zhou et al. (1993) introduced ‘universal I-PCR’ in which an antigen

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to be detected is coupled with antigen-specific primary antibody, followed by a species specific

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biotinylated detection antibody, streptavidin and the biotinylated DNA. The resulting method has been universally employed without any immunoglobulin (Ig) source or (sub) class restriction.

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Detection of a DNA-tag in I-PCR assays is examined by gel electrophoresis or real-time analysis of PCR products. Quantitative real-time I-PCR (RT-I-PCR), an evolution of I-PCR, has the

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potential to become the most analytically sensitive method for the detection of proteins (He and

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Patfield, 2015; Niemeyer et al., 2007).

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1.2 Different formats of I-PCR

There can be direct conjugation of antibody with the reporter DNA through covalent binding

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(Fig. 1b) via a chemical linker such as succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1carboxylate (SMCC) or N-succinimidyl-S-actyl-thioacetate (SATA, Liang et al., 2003; Fischer et

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al., 2007; Singh et al, 2015), which is quicker to perform than stretptavidin-biotin conjugates based I-PCR as there are less wash/incubation steps. Morin et al. (2011) and later Johnston et al.

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(2014) used a Tus-Ter complex for the detection of target antibodies by I-PCR using an LGcoated surface (Fig. 1b). The protein LG is a hybrid protein that consists of protein L from Peptostreptococcus magnus, which has an affinity to the Ig light chain and streptococcal protein G, which in turn has an affinity to the Fc region of IgG. The Tus protein is a monomeric replication terminator protein that binds tightly to short Ter DNA sequences present on Escherichia coli chromosome. An anti-target antibody fused to Tus was used for the detection of target antibody. The Ter DNA, which presents an affinity for the Tus protein, binds to anti-target antibody–Tus protein and was subsequently amplified. The Tus–Ter-lock I-PCR protocol uses the minimal number of washes, which is advantageous compared to the universal I-PCR with 5

ACCEPTED MANUSCRIPT streptavidin-biotin conjugates. The use of nanoparticles based I-PCR could further improve the detection limits by circumventing the background signals and reducing wash/incubation steps thus improving the assay (Chen et al., 2009; Perez et al., 2011). The large surface area of nanoparticles in comparison with that of microtitre plates/robostrips also allows higher and faster interaction between the capture antibody and respective antigen. In fact, the introduction of a liquid format by the use of magnetic beads (Barletta et al., 2009; Perez et al., 2013; Adams et al.,

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2012) has been a major breakthrough in devising I-PCR assays as it allows more thorough

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washing of the captured antigen and magnetic beads to decrease non-specific binding of reagents

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(Malou and Raoult, 2011; Perez et al., 2011) The quality of DNA-labeled affinity probes is crucial in DNA-assisted protein analyses of I-PCR reactions. Yan et al. (2014) described a

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universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing the unconjugated affinity reagents and remaining free oligonucleotides

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from conjugation reactions, which were used in proximity ligation assays in solution and in situ with augmented sensitivity and improved signal-to-noise ratios. A similar approach can also be

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used for I-PCR assays that can reduce background signals and further improve the sensitivity and specificity.

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Like ELISA, I-PCR can be performed in various formats depending on the goal of the experiment, for example, direct, indirect, sandwich and indirect sandwich I-PCR. Other formats

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include phage display-mediated I-PCR (PD-I-PCR), Magneto-I-PCR, Nanoparticle amplified IPCR (NPA-I-PCR) and immunoliposome-PCR (IL-PCR) assays (Fig. 1b, Mehta et al, 2014;

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Chang et al, 2016; Ryazantsev et al., 2016). PD-I-PCR involves a recombinant phage particle as a ‘ready reagent’ for I-PCR (Guo et al., 2006) as the surface displayed single chain variable

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fragment (scFv) and phage DNA themselves can serve directly as detection antibody and PCR template, respectively. In Magneto-I-PCR assay, the magnetic beads are captured using a magnetic plate (placed under the microtitre plate) periodically after each washing phase, which are then released from the magnetic force and are allowed to circulate freely in solution during incubation with antigen or antibody (Barletta et al, 2009; Wacker et al., 2007). In NPA-I-PCR, antigen is captured using antibody-functionalized magnetic beads (Perez et al., 2011, 2013). The gold nanoparticles (GNPs) are functionalized with the detection antibodies and thiolated DNA complementary to hybridized tag DNA. The magnetic bead–antigen complex is reacted with antibody–DNA-functionalized GNPs. The hybridized tag DNA (signal DNA) is released from 6

ACCEPTED MANUSCRIPT the GNPs by heating and is quantified by real-time PCR. On the other hand, IL-PCR method employs a liposome preparation incorporating reporter DNA encapsulated inside and a biotin labeled polyethylene glycol (PEG) phospholipid conjugate as a detection reagent in the outer surface of a liposome, which is coupled to real-time PCR for antigen detection (He et al., 2012). I-PCR assays have been widely used for the detection of several biological molecules such as protooncogenes, cytokines, T-cell receptors, biomarkers for Alzheimer’s disease and can be

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adopted as a novel diagnostic tool for infectious and non-infectious diseases (Malou and Raoult,

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2011; Niemeyer et al., 2005; Mehta et al., 2014; Chang et al, 2016; Ryazantsev et al., 2016). This

antibodies in body fluids of TB patients by I-PCR assays.

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2. Antigen detection

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review is primarily focused on the detection of potential mycobacterial antigens and circulating

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Several mycobacterial antigens including immunodominant antigen 85B (Ag85B, Rv1886c) and PstS1 (Rv0934) as well as regions of differences (RD)1 and RD2 encoded proteins such as

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ESAT-6 (Rv3875), CFP-10 (Rv3874), MPB-64/MPT-64 (Rv1980c) and CFP-21 (Rv1984c) have been detected by I-PCR in body fluids of TB patients, which revealed better results than ELISA

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(Mehta et al., 2012a, Mehta et al., 2016; Sharma et al., 2017). The starting material for the purified or recombinant antigens used in these studies was from M. tuberculosis H37Rv and there

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was no strain variation for setting up such assay systems. Bekmurzayeva et al. (2013) and later Tucci et al. (2014) have meticulously reviewed the detection of array of antigens including

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ESAT-6, CFP-10, MPT-64, PstS1, HspX (Rv2031c), Ag85 complex, lipoarabinimannan (LAM), etc. for the serodiagnosis of TB by ELISA and immunochromatographic assay, which leads to

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variable results. Among these, antigen 85 (Ag85) complex is the major culture filtrate protein of M. tuberculosis comprised of three related proteins, Ag85A, Ag85B, and Ag85C expressed in a 3:2:1 ratio of B:A:C, but the ratio varies in response to environmental changes (Ronning et al., 2004). Within Ag85 complex, Ag85B is a potential biomarker for TB diagnosis since this is the most abundant secreted protein (40%) of M. tuberculosis (Therese et al, 2012), which posses mycolyl transferase activity thus leading to the formation of 6,6′-trehalose monomycolate and cord factor (6,6′-trehalose dimycolate) that are essential for the cell wall biosynthesis (Aghababa et al., 2011). Cord factor is considered as a genuine virulence factor of M. tuberculosis, which is crucial for the survival of bacteria inside the macrophages as it prevents the fusion of 7

ACCEPTED MANUSCRIPT phagosomal vesicles with the lysosomes within the cultured macrophages (Hunter et al., 2006; Lang, 2013). LAM, a lipopolysaccharide of mycobacterial cell wall is released from metabolically active bacteria and is present in individuals with active TB, whose primary function is to inactivate macrophages and scavenge oxidative radicals (Tabarsi et al., 2017). Unlike Ag85B, PstS1 is highly M. tuberculosis specific and is absent in most of the nontuerculous mycobacteria (NTM) and occurs in lesser amounts in M. bovis bacillus Calmette-

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Guérin (BCG) strains (Freeman et al., 1999; Goel et al., 2012). The secreted PstS1 protein is

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produced by actively growing cultures of M. tuberculosis and is considered as a marker for

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active mycobacterial replication in contrast to ESAT-6 and 14 kDa (Rv0251c) detection (Silva et al., 2003; Haldar et al., 2012). On the other hand, HspX plays a crucial role in the maintenance of

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long-term viability of bacteria during latent, asymptomatic infection (Atieh et al, 2016), which elicits specific humoral and cellular immune responses in individuals with active as well as latent

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TB infection (Haldar et al., 2012; Silva et al., 2014). However, PstS1 reacts with sera from human patients with multibacillary or advanced PTB but is poorly recognized in sera from

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patients with low bacillary counts in sputum and from asymptomatic infected persons by ELISA, whereas an inverse correlation has been found between the bacterial load and ESAT-6

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specific humoral response (Abebe et al., 2007; Pathan et al., 2001; Silva et al., 2003). ESAT-6 and CFP-10 are the most popular antigens of M. tuberculosis complex that are

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co-transcribed to form a tight 1:1 heterodimer complex (Renshaw et al., 2002, Van Ingen et al., 2009), which are implicated in virulence mechanisms and can induce apoptosis of macrophages

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(Guo et al., 2012). Both of these proteins are important stimulator of T-cells and are widely employed as eliciting agents in IGRAs (Song et al., 2012). Similar to RD1 encoded proteins,

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MPT-64 and CFP-21 are M. tuberculosis specific and are absent in most of the BCG strains, M. leprae and NTM (Kanade et al, 2012). Both of these proteins also play a key role in virulence mechanisms and induce delayed-type hypersensitivity responses in sensitized guinea pigs (West, et al., 2008; Kalra et al., 2010b). MPT-64 is secreted in significant amounts during early phase of growth and decreases with long cultivation of bacteria (Kanade et al, 2012). Interestingly, CFP21 has been shown to posses cutinase, esterase and lipolytic activities (Grover et al., 2006; Parker et al., 2007).

2.1 Antigen detection by I-PCR 8

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Representative of positive TB samples evaluated by I-PCR for antigen detection are shown (Fig. 2a). The sample showing an amplification product of 110 bp on gel electrophoresis was considered positive for the presence of mycobacterial antigens (Mehta et al., 2016). An indirect sandwich I-PCR with streptavidin-biotin conjugates was developed for the detection of RD1 and RD2 antigens in sputum samples of PTB patients (Mehta et al., 2012a). The highest sensitivity

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was observed for the detection of a cocktail of four RD proteins (ESAT-6, CFP-10, MPB-64 and

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CFP-21) in both smear-positive (77%) and smear-negative (68%) PTB patients. However, the

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differences in sensitivities of individual RD proteins and cocktail of four RD proteins (Fig. 2b) were not found to be statistically different (P > 0.05) for both smear-positive and smear-negative

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PTB patients. Furthermore, a specificity of 85-90% was observed for all the individual proteins, which was not jeopardized by detecting a cocktail of four RD proteins by I-PCR. EPTB samples

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such as cerebrospinal fluids, ascitic fluids and pleural fluids were also analyzed for the presence of RD proteins (Fig. 2c). Notably, detection of cocktail of four RD proteins in EPTB samples by

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I-PCR assay could reveal an enhanced sensitivity (54% versus 31%) as compared to ELISA (Kalra et al., 2010a) without compromising the specificity (90-95%). Table 1 summarizes the

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serodiagnostic potential of I-PCR assays for the detection of mycobacterial antigens and antibodies in body fluids of TB patients and its comparative evaluation with ELISA.

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I-PCR assay based on streptavidin-biotin system has limitations as there are more incubation/wash steps, which can also lead to background signals (Malou and Raoult, 2011;

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Mehta et al., 2014). Therefore, the amino-modified reporter DNA was covalently attached to the dithiothreitol (DTT)-reduced antibody via SMCC to develop indirect sandwich I-PCR for the

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detection of Ag85B in TB patients, which displayed a higher sensitivity of 95.2-100% in smearpositive, culture-positive PTB cases (Singh et al., 2015; Mehta et al., 2016) as compared to ELISA. While broadly categorizing sputum samples on basis of smear microscopy, sensitivities of 83.3% and 75% were observed in smear-positive and smear-negative PTB patients with a specificity of 92.8% by I-PCR for Ag85B detection, whereas sensitivities of 85.7% and 62.8% were observed in smear-positive confirmed and smear-negative clinically suspected EPTB patients with a specificity of 90% (Fig. 2d-e; Table 1). High sensitivity and specificity exhibited with Ag85B detection by I-PCR was also validated in a higher number of PTB (n = 182) and EPTB (n = 105) samples, which showed reproducible results (Singh et al., 2015). Wallis et al. 9

ACCEPTED MANUSCRIPT (1998) previously differentiated persister and nonpersister bacteria based on Ag85 complex detection in sputum samples of PTB patients (on therapy) by ELISA thus indicating the presence of M. tuberculosis. Similarly, Phunpae et al. (2014) described the detection of Ag85 complex by ELISA for the identification of M. tuberculosis, when sputum samples of PTB patients were inoculated into liquid medium and the culture filtrates were collected at different days for Ag85 complex detection, which revealed somewhat faster results than the standard culture method. On

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the contrary, detection of Ag85B by I-PCR yielded rapid results with higher sensitivity (Mehta et

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al., 2016; Singh et al., 2015). Few positive samples missed by I-PCR could be attributed due to

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either very low undetectable Ag85B level in the samples or antigen sequestering within antigenantibody complexes (Haldar et al., 2012). While detecting Ag85B from pleural fluids (n= 64) for

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the diagnosis of TB pleuritis, a sensitivity of 76.5% has been demonstrated by I-PCR (Singh et al., 2015). Based on meta-analysis from six studies (598 samples) on Xpert assay, a lesser

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sensitivity of 21.4% has been reported for the diagnosis TB pleuritis, using composite reference standard (CRS) as the reference standard, whereas a sensitivity of 46.4% was observed from 14

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studies (841 samples), using culture as the reference standard (Denkinger et al., 2014). Therefore, the WHO has not recommended the use of Xpert assay for the diagnosis of TB

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pleuritis, though the same assay has been recommended for the diagnosis of PTB, TB lymphadenitis and TB meningitis. Hence, it has been suggested to compare I-PCR versus Xpert

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assay on the same specimens to ensure the efficacy of I-PCR test for the competent diagnosis of TB pleuritis.

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Attempts were made to further enhance the sensitivity of I-PCR assay based on SMCC for the detection of a cocktail of mycobacterial Ag85B, ESAT-6 and cord factor in body fluids of

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TB patients. Interestingly, the detection of Ag85B was found to be superior to the detection of a cocktail of antigens (Ag85B, ESAT-6 and cord factor) followed by cord factor and ESAT-6 (Fig. 1d), taking CRS as the reference standard (Mehta et al., 2016). While considering culture as the sole criterion in diagnosing PTB patients, detection of Ag85B by I-PCR also exhibited better positivity than the detection of a cocktail of antigens in culture-positive confirmed (95.1%) as well as culture-negative suspected PTB cases (66.6%). Similarly, in clinically suspected EPTB patients, better sensitivity was observed with Ag85B detection (62.8%) in comparison to cocktail (Fig. 1e), whereas a higher sensitivity of 85.7% was observed for both Ag85B and cocktail detection in confirmed EPTB cases. The reason for better positivity with Ag85B by I-PCR could 10

ACCEPTED MANUSCRIPT also be the choice of Ag85B protein as a biomarker since it is the most abundant secreted protein of M. tuberculosis (Singh et al., 2015) and the use of polyclonal antibody instead of monoclonal antibody in indirect sandwich I-PCR as that has higher avidity and broader range of epitope recognition capacity (Fischer et al., 2007). Similar to these findings, Kumar et al. (2010) earlier demonstrated lesser sensitivity in PTB patients using a cocktail of Ag85 complex, ESAT-6 and CFP-10 by ELISA in comparison to individual antigens. On the contrary, while detecting a

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cocktail of RD proteins such as ESAT-6, MPT-64, etc. in TB patients by I-PCR, the sensitivity

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was enhanced in comparison to individual RD protein (Fig. 2b; Mehta et al., 2012a). However,

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Shekhawat et al. (2016) documented lesser sensitivity with the combined use of host heat shock proteins (Hsp 25, Hsp 70, etc.) for the diagnosis of TB meningitis by ELISA in comparison to

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individual Hsps. The lesser sensitivity with a cocktail of Ag85B, ESAT-6 and cord factor in comparison to Ag85B could be due to competition of different antigens in TB samples with the

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capture antibodies, steric hindrance or masking of some of the dominant epitopes in cocktail detection (Kumar et al., 2010; Mehta et al., 2016). Furthermore, lesser positivity was also

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observed for cord factor detection in body fluids of PTB (60%) as well as EPTB (42.8%) cases by I-PCR in comparison to Ag85B detection but with good specificity (92.8-93.3%), however,

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the detection of cord factor has advantage of using the heat-killed (autoclaved) samples and that leads to minimal manipulation of biohazard (Mehta et al., 2016).

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Notably, the specificity (90-95.2%) was not jeopardized by detecting a cocktail of antigens (Ag85B, ESAT-6 and cord factor) in respiratory disease/non-TB controls with I-PCR

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(Mehta et al., 2016). Respiratory disease control specimens included sputum samples from patients suffering from asthma, non-TB pneumonia and chronic obstructive pulmonary disease

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(COPD). On the contrary, non-TB control specimens included pleural fluids from patients suffering from lung cancer, kidney cancer, colon cancer, etc. as well as from trauma patients due to rib fractures and postoperative abdominal surgery. Pleural fluids were also collected from individuals suffering from renal failure due to nephrotic syndrome and pancreatitis as controls. Similarly, ascitic fluids were collected from individuals suffering from non-TB gastrointestinal diseases such as stomach cancer, liver diseases and lymphomas as controls. However, few false positive results, that were noticed for Ag85B, ESAT-6, cord factor and cocktail detection, in controls suggest that I-PCR assay is also vulnerable to sensing non-specific signals and the sample matrix effect, which may be improved by the use of GNPs/magnetic beads based I-PCR 11

ACCEPTED MANUSCRIPT assays (Malou and Raoult, 2011; Mehta et al., 2014). Since Ag85B and cord factor are present in all mycobacteria including BCG strains and the NTM, I-PCR based on Ag85B and cord factor detection might give false-positive results with other mycobacterial infections as the prevalence rate of NTM from India has been reported ranging from 0.5% to 8.6% (Jain et al., 2014). However, the presence of Ag85B and cord factor in BCG strains and the NTM would unlikely influence the analysis of I-PCR results, as almost all the controls were vaccinated with BCG and

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exposed to the same environment (Singh et al., 2015).

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2.2 Antigen detection by RT-I-PCR

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An indirect RT-I-PCR based on streptavidin-biotin conjugates has recently been developed for the quantitative detection of PstS1 in body fluids of TB patients (Sharma et al., 2017). The

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threshold cycle (Ct) was determined by setting fluorescence in the exponential phase of the amplification curves, reading out the fractional cycle number at which the amplification curve

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crossed the threshold (Fig. 3a) and the standard curve was plotted between the Ct values and log PstS1 concentration (Fig. 3b). The final detection range of purified PstS1 was 10 ng/mL to 100

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fg/mL by RT-I-PCR and that was much larger than ELISA (1 μg/mL to 1 ng/mL). The detection limit of PstS1 was determined to be ~ 40 fg/mL by RT-I-PCR, which was ~ 2.5 X 104-fold lower

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than ELISA. RT-I-PCR could detect a higher concentration of PstS1 and in a higher number of TB samples in comparison to ELISA. In positive TB samples, a wide dynamic range of 10

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ng/mL to 1 pg/mL of PstS1 was detected by RT-I-PCR. As compared to ELISA, the reasonable higher number of positive cases were recognized from both smear-positive (78.9%) and smear-

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negative (64.9%) PTB cases by RT-I-PCR with a specificity of 90.5% (Table 1), taking CRS as the reference standard. Considering culture as the sole criterion in diagnosing PTB cases, RT-IPCR also displayed better sensitivity than ELISA in both culture-positive confirmed (81.6%) and culture-negative suspected cases (56.3%). Similarly, better sensitivities were observed with RTI-PCR in smear-positive confirmed (70%) and smear-negative clinically suspected EPTB cases (62.5%) with a specificity of 90% (Sharma et al., 2017). The quantification of a wide dynamic range of PstS1 by RT-I-PCR in bodily fluids of TB patients probably indicates the multiplication of M. tuberculosis inside the host cells, which might have potential implications to study the disease progression, clinical symptoms and response to anti-tubercular therapy (ATT) in 12

ACCEPTED MANUSCRIPT different clinical stages. Notably, Shi et al., (2004) earlier documented that the arrest of M. tuberculosis growth in mouse lung was accompanied by several-fold decrease in mRNA levels encoding the PstS1 and Ag85 complex, whereas esat-6 mRNA levels were high throughout infection thus implying that PstS1 and Ag85 complex secretion was associated with multiplying bacteria inside the host cells, and the multiplying and nonreplicating tubercle bacilli have different antigen compositions. However, further work is recommended to enhance the

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sensitivity of RT-I-PCR in TB patients by detecting a cocktail of antigens including PstS1.

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Similar to this study, there are reports for the detection of other bacterial and viral

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antigens, for example, C. botulinum neurotoxin A, Cry1Ac protein of B. thuringiensis var. kurstaki, Group A S. pyogenes and H5N1 avian influenza virus by I-PCR based on SMCC with

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several-fold magnitude below the detection limit of ELISA (Allen et al., 2006; Deng et al., 2011; Mehta et al., 2014). Instead of using SMCC, SATA was employed for the covalent binding of

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reporter DNA with the antibody by Fischer et al. (2007) for the detection of S. aureus enterotoxin (SE)A and SEB by RT-I-PCR. Both SMCC and SATA have also been used for the

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detection of trace-levels of chloramphenicol in milk by RT-I-PCR (Tao et al., 2014). Using streptavidin-biotin system, the detection limit of Shiga toxin 2 (Stx2) was determined to be 0.1

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pg/mL in buffer by RT-I-PCR with a wide dynamic range of 10–100,000 pg/ml, which was 104fold more sensitive than ELISA (He et al., 2011). In addition, Stx2 and its variants could be

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detected in environmental samples contaminated with soil, faeces and water samples, whereas ELISA could not detect Stx2 in those contaminated samples. The detection of recombinant

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hepatitis B surface antigen and HIV p24 protein from human serum and plasma by PD-IPCR/Magneto-I-PCR has also been documented (Wacker et al., 2007; Barletta et al., 2009;

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Monjezi et al., 2013). In addition, the ultralow detection of environmental pollutants and carcinogens such as 17β-estradiol, pyrene and related polycyclic aromatic hydrocarbons in water samples has been demonstrated by RT-I-PCR (Gaudet et al., 2015; Meng et al., 2016), which showed several-fold higher sensitivity than ELISA. Likewise for protein detection, the proteolytic activity of anthrax and botulinum toxins has recently been documented by Kolesnikov et al. (2016) with RT-I-PCR at sub-pg levels using composite probes consisting of covalent peptide-DNA conjugate and noncovalent protein-aptamer assembly to assay anthrax and botulinum toxins, respectively.

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ACCEPTED MANUSCRIPT 3. Antibody detection

In addition to antigen detection, circulating antibodies to important mycobacterial components have been detected in bodily fluids of TB patients by I-PCR assays. Several studies have previously indicated that the pattern of antigen reactivity to various antibodies in serum varied greatly from patient to patient and no antigen alone can perform significantly for TB diagnosis

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(Abebe et al., 2007; Imaz et al., 2008; Khalid et al., 2016). Therefore, a cocktail of M.

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tuberculosis-specific RD1 and RD2 proteins, that is, ESAT-6, CFP-10, MPT-64 and CFP-21 as

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well as a cocktail of Ag85B, ESAT-6 and cord factor were used to design I-PCR assays for detecting circulating antibodies to minimize the possible heterogeneity of antigen recognition in

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TB patients, which revealed better results than ELISA. There are many reports available to detect circulating antibodies to mycobacterial antigens such as ESAT-6, MPT-64, Ag 85 complex,

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PstS1, cord factor, LAM, TB16.3 (Rv2185c) etc. by ELISA and immunochromatographic assay for the serodiagnosis of TB (Abebe et al., 2007; Imaz et al., 2008; Kalra et al., 2010a., Liu et al.,

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2016; Ben-Selma et al., 2011), which leads to variable results. In fact, all the commercial tests based on antibody detection failed due to inconsistent and imprecise results, therefore, the WHO

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has strongly recommended against the use of any of these tests, but also stressed to design new antibody-detection tests with improved accuracy (Steingart et al., 2011) An indirect I-PCR with

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streptavidin-biotin system has been developed to detect anti-RD antibodies, that is, anti-ESAT-6, anti-CFP-10, anti-MPB-64 and anti CFP-21 antibodies in sera samples of PTB patients (Mehta et

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al., 2012a). The sensitivities for individual anti-RD antibodies and their cocktail by I-PCR are shown (Fig. 4a). Interestingly, detection of cocktail of four anti-RD antibodies exhibited the

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highest sensitivity for both smear-positive (77%) and smear-negative (59%) PTB cases in comparison to individual anti-RD antibodies by I-PCR (Fig. 4a). Furthermore, specificities of 90-95% and 85%, were observed for individual anti-RD antibodies and cocktail, respectively, by I-PCR. However, I-PCR assays for the detection of anti-RD antibodies in body fluids of EPTB patients could not be performed as the sample volumes were not adequate (Mehta et al., 2012a). In a similar manner to antigen detection, the amino-modified reporter DNA was covalently attached to DTT-reduced antibody via SMCC to design indirect I-PCR for the detection of a cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies in sera samples of TB patients (Singh et al., 2016). The sensitivities of 89.5% and 77.5% were observed 14

ACCEPTED MANUSCRIPT for the detection of a cocktail of antibodies (followed by anti-cord factor, anti-Ag85B and antiESAT-6 antibodies) in smear-positive and smear-negative PTB cases, respectively, by I-PCR with a specificity of 90.9% in respiratory disease controls and 93.3% in healthy controls (Fig. 4b, Table 1). Unlike anti-RD antibodies detection, significant differences (P < 0.001 - 0.05) were also observed in sensitivities between the individual antibodies and their cocktail (anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies) for both smear-positive and smear-negative PTB

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cases. On the other hand, in EPTB cases, sensitivity and specificity of 77.5% and 92-93.3% was

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observed by I-PCR for the detection of a cocktail of antibodies (Fig. 4c, Singh et al., 2016). The

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extensive extracellular bacterial replication in lung cavities and the continued release of antigens was possibly enhanced in the lung cavitary environment thus leading to high antibody response

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in smear-positive PTB patients for the detection of a cocktail of antibodies by I-PCR. The paucibacillary and often walled off EPTB lesions might be different in producing lesser amount

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of antigen and thus could elicit lesser antibody response (Kaushik et al., 2012). As observed with healthy/respiratory disease controls, few false positive results were observed in sera of non-TB

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controls by I-PCR for the detection of individual or cocktail of antibodies by I-PCR thus leading to a specificity of 88-92%. These findings demonstrate that I-PCR for antibody detection is also

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vulnerable to sense non-specific background signals and the sample matrix effect. Interestingly, detection of antibodies in pleural/ascitic fluids of TB patients by I-PCR

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showed lesser sensitivities in comparison to sera samples, though relatively higher specificity (96%) was observed. However, similar to sera samples, the highest sensitivity (70%) was

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observed with the detection of a cocktail of antibodies (anti-Ag85B, anti-ESAT-6 and anti-cord factor) in pleural/ascitic fluids (Fig. 4c, Singh et al., 2016). Similar findings were reported by

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Wankhade et al. (2012) with SEVA TB ELISA. On the contrary, a higher sensitivity of 75.4% was attained earlier by Sumi and Radhakrishnan (2010) in pleural fluids with a cocktail of antiESAT-6, anti-Plc1 (membrane-associated phospholipase C1, Rv2351c), anti-HspX and antiTB8.4 (Rv1174c) antibodies. Almost similar sensitivity (76%) was achieved with the combined detection of anti-MPT-64 and anti-MT-10.3 (Rv3019c) IgA antibodies in pleural fluids by ELISA (Kaisermann et al., 2005), which was further raised to 81.4% using a fusion protein MPT-64-MT-10.3 (Araujo et al., 2010). However, a sensitivity of 83.3% was documented in sera samples of pleural TB patients by I-PCR for the detection of a cocktail of anti-Ag85B, antiESAT-6 and anti-cord factor antibodies (Singh et al., 2016). 15

ACCEPTED MANUSCRIPT Similar to this study, there are reports for the better detection of circulating antibodies in other viral and bacterial infections by I-PCR as compared to ELISA. For example, Mweene et al. (1996) previously demonstrated antibodies to bovine herpes virus type 1 by I-PCR in experimentally infected calf/rabbit sera samples with a 105-fold higher sensitivity than ELISA. Anti-Coxiella burnetii IgM antibodies were detected by Malou et al. (2012) for the diagnosis of Q fever, which revealed the highest sensitivity with I-PCR (87%), followed by PCR (58%),

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ELISA (38%) and immunofluorescent antibody test (32%). In a similar manner, Halpern et al.

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(2013) documented the superiority of I-PCR using magnetic beads in diagnosing Lyme disease

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for the detection of anti-Borrelia burgdorferi antibodies in experimentally infected mice at day 7-11 post-inoculation (p.i.) that could be detected at day 14 p.i. by ELISA and day 21p.i.by

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Western blotting. The serum IgE antibodies specific to Bermuda grass allergans have also been detected by I-PCR, which showed 100-fold enhancement in sensitivity over ELISA (Rahmatpour

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et al., 2017).

In addition, paired sample analyses involving antibody detection in serum and testing the

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presence of RD antigens in sputum samples of the same individual demonstrated an improvement in the recognition of PTB patients (Fig. 4d, Mehta et al., 2012a). Interestingly,

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combining the two tests did not significantly affect the specificity of either of the single tests. Previous study using RD antigen-based ELISA as well as other studies showed a marked

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increase in the sensitivity of diagnostic assays for TB using paired samples for detecting either antibodies in both the samples or antigens and antibodies in different samples (Singh et al., 2003;

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Stavri et al., 2003; Kalra et al., 2010a). Notably, the sensitivity of RD multi-antigen cocktail IPCR assay was significantly enhanced by paired sample analysis in both smear-positive (91%)

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and smear-negative (72%) PTB patients but with a specificity of 85% (Mehta et al., 2012a). Overall, both antigen and antibody detection showed encouraging results. Among array of antigens and antibodies tested, detection of Ag85B protein and cocktail of anti-Ag85B, antiESAT-6 and anti-cord factor antibodies in bodily fluids of PTB and EPTB patients by I-PCR revealed the highest sensitivities with good specificity values, but that did not differ significantly with the detection of corresponding anti-Ag85B antibody and cocktail of antigens (Fig. 1, 3). However, direct detection of M. tuberculosis antigens allows specific diagnosis of active disease independent of host’s immune response (Flores et al., 2011). Antigens that are shed from M. tuberculosis in infected tissues can be present in bodily fluids where from they reach the blood 16

ACCEPTED MANUSCRIPT circulation and are eliminated in urine. Urine is easy to collect from both adults and children that could facilitate TB diagnosis in HIV co-infected and HIV uninfected TB patients thus preventing the use of aggressive invasive methods for collecting the samples (Tucci et al., 2014). The detection of LAM as a biomarker in urine samples of HIV co-infected and HIV uninfected TB patients by ELISA as well as lateral flow immunochromatographic test has been established yet both of the assays showed lesser sensitivity for HIV uninfected TB patients (Flores et al., 2011;

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Nakiyingi et al., 2014). Therefore, it is recommended to enhance the sensitivity while

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maintaining high specificity for the detection of mycobacterial enriched antigens such as Ag85B,

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PstS1, LAM, etc. in urine samples by isolating exosomes followed by I-PCR test. In fact, the TB community has expressed the need for several additional TB diagnostic

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tests (Houben et al., 2014). Sensitivity and specificity are crucial factors for immunoassays including I-PCR. In a consensus meeting, WHO (2014) has described the optimal sensitivities

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and specificities of high-priority target product profiles (TPPs) to design new TB diagnostics as rule-in or rule-out tests. The aim of TPPs is to design POC non-sputum based test capable of

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detecting all forms of TB (PTB, EPTB, childhood TB and HIV-coinfected TB) by identifying appropriate biomarkers as well as to design POC triage test which can be used for first-contact

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health-care individuals and POC sputum-based test to replace smear microscopy. It is recommended that the rapid sputum-based test replacing smear microscopy at the microscopy-

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centre level should have a sensitivity of > 95% for a single test for detecting PTB when compared with culture (should be > 99% for smear-positive cases and > 68% for smear-negative

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cases), whereas specificity should be ≥ 98%. In this study also, high sensitivities of 95.2-100% and 95% were observed in smear-positive (n = 48) and smear-negative (n = 39) sputum samples

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of PTB cases, respectively by I-PCR based on Ag85B detection, using culture as the reference standard. However, data for culture test and Xpert assay were not available for many samples of this study as these tests are not performed in routine use in India. While broadly categorizing sputum samples into smear-positive (n = 170) and smear-negative (n = 72) PTB cases, sensitivities of 80-83% (83.3-85% for smear-positive cases and 77-80% for smear-negatives cases) and specificities of 90.3-92.8% in respiratory disease controls (n = 217) were observed by I-PCR based on Ag85B detection, using CRS as the reference standard. In a similar I-PCR reaction, sensitivities of 84-85.7% and specificities of 90-92% were observed in pleural/ascitic fluids of smear-positive (n = 32) confirmed EPTB cases and non-TB controls (n = 80), 17

ACCEPTED MANUSCRIPT respectively. Though I-PCR assays are robust and well-established for ultralow detection of target antigens in biological samples, these assays are also vulnerable to sense non-specific signals thus compromise over the accuracy. Furthermore, in many resource-limited settings, TB diagnosis is made only by a single test. To secure an appropriate positive predictive value, specificity is more important than sensitivity in some cases, which may be improved by the use of nanoparticles based test. It would be worthwhile to design I-PCR test using appropriate

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biomarkers for non-sputum samples such as blood, urine, saliva, oral transudates and exalted air

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(collected by non-invasive methods) of PTB and EPTB patients with enhanced sensitivity and

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specificity to meet the WHO guidelines, using culture, Xpert assay or CRS as the reference

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standard.

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4. Merits

Detection of mycobacterial antigens and antibodies in body fluids by I-PCR assays offers a

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robust approach that reveals rapid results with good diagnostic accuracy for an early diagnosis of TB, which shows superiority over ELISA. The most benefitted groups would be smear-negative

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PTB and paucibacillary EPTB cases that are otherwise quite difficult to diagnose. The quantitative detection of a wide dynamic range of mycobacterial antigens in body fluids by RT-I-

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PCR may reflect the progression of disease and response to ATT.

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5. Demerits

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The major problem of I-PCR assays for TB diagnosis remains the high background noise due to non-specific binding and the sample matrix effect thus compromising the accuracy of test as well as many steps in the protocol. This might be circumvented by the exploitation of a liquid format using magnetic beads/GNPs based I-PCR and that can also establish an automated one-step IPCR and thus a reduction of the overall duration of assay.

6. Conclusion

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ACCEPTED MANUSCRIPT This review has described the utility of serodiagnostic potential of I-PCR for the detection of mycobacterial antigens and antibodies in body fluids of TB patients. Among the several antigens detected by I-PCR, Ag85B showed the highest sensitivity, followed by the detection of a cocktail of Ag85B, ESAT-6 and cord factor. On the other hand, detection of a cocktail of anti-Ag85B, anti-cord factor and anti-ESAT-6 antibodies in sera of TB patients revealed better sensitivity in comparison to the detection of individual antibodies. Detection of Ag85B protein and cocktail of

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antibodies with I-PCR also revealed good specificity. Though both antigen and antibody

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detection showed promising results, antigen detection is considered more accurate than antibody

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detection. Hence, further studies were conducted for the quantitative detection of PstS1 in body fluids by RT-I-PCR, which may monitor the dynamics of disease. However, further work is

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warranted to design a cost-effective I-PCR test with further improved accuracy so that it can be

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included in the diagnostic panel for routine use especially in resource-poor settings.

Acknowledgements

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This work was financially supported by DBT (BT/PR8054/MED/29/710/2013), New Delhi, DST (DST/SSTP/Haryana/2012–13/12th Plan/39G), New Delhi and DBT-Builder Programme

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(BT/PR4329/INF/22/144/2011), New Delhi. BD acknowledges DBT (DBT/JRF/14/AL/243),

Conflict of interest

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New Delhi for providing Junior Research Fellowship.

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The authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT Zhou, H., Fisher, R. J., Papas, T. S., 1993. Universal immuno-PCR for ultra-sensitive target

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1 (a) Evolution of I-PCR in typical sandwich format: I-PCR is similar to ELISA except at the terminal step, there is amplification of a reporter DNA by PCR (Zhou et al., 1993). (b) Different formats of I-PCR: (i) Chemical conjugation of detection antibody and DNA with succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC)/N-succinimidyl-Sactyl-thioacetate (SATA) (Liang et al., 2003; Fischer et al., 2007) (ii) Tus-Ter system for

T

detection of antibodies: LG protein is coated on a microtitre plate and target antibody binds to it,

IP

which in turn binds to anti-target antibody fused with Tus protein. Tus protein binds to Ter DNA

CR

sequences and DNA is amplified by PCR/Real-time PCR (Morrin et al., 2011) (iii) Phage display-mediated I-PCR (PD-I-PCR): DNA and single chain variable fragment (scFv) both are

US

carried by recombinant phage. The scFv is attached to antigen and phage DNA is released on heating, which is detected by PCR/Real-time PCR (Guo et al., 2006) (iv) Magneto-I-PCR:

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Antigen is attached to the capture antibody on magnetic beads (by using a magnetic plate) in wells of microtitre plate. After that, sandwich I-PCR format is followed and DNA is amplified

M

by PCR/Real-time PCR (Barletta et al., 2009). (v) Nanoparticle-amplified I-PCR (NPA-I-PCR): Magnetic beads are attached to capture antibody, which binds to antigen. After that,

ED

functionalized GNP probes are added to form sandwich immunocomplexes; the signal DNA is released on heating, which is amplified by PCR/Real-time PCR (Perez et al., 2011). (vi)

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Immunoliposome-PCR (IL-PCR): Antigen is sandwiched between capture antibody and biotinylated detection antibody. Neutravidin is used as a bridge between biotinylated detection

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antibody and biotin-labeled PEG polymer with the phospholipid component (not shown) embedded in the outermost bilayer leaflet of the liposome. Encapsulated DNA reporters are seen

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inside the liposome. Liposomes are ruptured and DNA is amplified by PCR/Real-time PCR (He et al., 2012).

Fig. 2 (a) Agarose gel analysis of I-PCR product (110 bp) for representative clinical TB samples: lane M, represents 100-bp molecular marker; lanes 1-3, positive PTB samples; lanes 4-6, respiratory disease controls; lanes 7-9, positive EPTB samples; lanes 10-14, non-TB samples controls; lane 15, ‘no template DNA’; lane 16, ‘PCR grade water’; lane 17, negative control (‘no antigen’); and lane 18, positive control (‘cocktail of three antigens’) (Mehta et al., 2016). (b) Sensitivities of antigen detection in sputum samples of smear-positive (n = 40) and smearnegative (n = 25) PTB patients by indirect sandwich I-PCR. COMB. = Combination of four RD 30

ACCEPTED MANUSCRIPT antigens used; ANY = positive for the presence of any of four RD antigens. positive cases and

represents smear-

represents smear-negative cases (Mehta et al., 2012a). (c) Recognition of

EPTB patients (n = 35) on the basis of antigen detection by I-PCR in extrapulmonary fluids. represents EPTB. Chi-square analysis with Yates correction for comparing 2 antigens showed significantly higher sensitivity of the combination as compared to CFP-10 (* indicates P < 0.05) (Mehta et al., 2012a). (d) Comparison of individual antigens and cocktail in PTB samples:

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sensitivities of antigen detection in smear-positive (n = 36) and smear-negative (n = 24) PTB by

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ELISA and I-PCR based on individual antigens and their cocktail. Differences between the

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sensitivities of ESAT-6, cord factor, cocktail and Ag85B were found to be statistically significant by group analysis using chi-square test (* indicates P < 0.01 for ESAT-6, $ indicates

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P < 0.01 for cord factor and # indicates P < 0.05 for cocktail of antigens) (Mehta et al., 2016) (e) Comparison of individual antigens and cocktail in EPTB samples: sensitivities of antigen

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detection in EPTB patients (n = 42) by ELISA and I-PCR based on individual antigens (ESAT-6, Ag85B, cord factor) and their cocktail. Differences between the sensitivities of ESAT-6, cord

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factor, the cocktail and Ag85B were found to be statistically significant by group analysis using chi-square test (* indicates P < 0.001 for ESAT-6, $ indicates P < 0.001 for cord factor and #

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indicates P < 0.05 for the cocktail of antigens) (Mehta et al., 2016) Fig. 3 (a) Amplification curves for various dilutions of purified PstS1, which ranged from 10

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ng/mL to 100 fg/mL; Background control (no reporter DNA); Negative control (no PstS1). The experiment was performed in duplicate and one of the representative curves has been shown. (b).

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Standard curve of purified PstS1 in coating buffer by RT-I-PCR and ELISA. The correlation coefficient of RT-I-PCR was 0.98 and the corresponding regression equation was Ct= −1.43× log

2017).

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(conc.) + 35.15. The mean Ct and OD values were derived from two replicates (Sharma et al.,

Fig. 4 (a) Sensitivities of antibody detection in smear-positive (n = 65) and smear-negative (n = 37) PTB patients by I-PCR based on RD antigens and their combination. COMB. = Combination of four RD antigens used; ANY = positive for antibody detection using either of four RD antigens.

represents smear-positive cases and

represents smear negative cases. Differences

between the sensitivities of individual antigens and the combination were found to be statistically significant by group analysis using chi-square test (P < 0.0001 for smear-positive cases and P < 0.05 for smear-negative cases). Chi-square analysis with Yates correction for comparing 2 31

ACCEPTED MANUSCRIPT antigens showed significantly higher sensitivity of the combination as compared to all individual antigens in the case of smear-positive PTB patients (# indicates P < 0.001 for ESAT-6, + indicates P < 0.01 for CFP-10, and $ indicates P < 0.0001 for both CFP21 and MPT- 64) and to all antigens except MPT-64 in case of smear-negative group (* indicates P < 0.05) (Mehta et al., 2012a). (b) Sensitivities of antibody detection in smear-positive (n = 48) and smear-negative (n = 40) PTB patients by ELISA and I-PCR based on individual antibodies and their cocktail.

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Differences between the sensitivities of individual antibodies and their cocktail were found to be

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statistically significant by group analysis using chi-square test (* indicates P < 0.001 for ESAT-

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6, $ indicates P < 0.001 for Ag85B, and # indicates P < 0.001 for cord factor) (Singh et al., 2016). (c) Sensitivities of antibody detection in sera and pleural/ascitic fluids from the same

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EPTB (n = 40) patients by ELISA and I-PCR based on detection of individual antibodies (antiESAT-6, anti-Ag85B, anti-cord factor antibodies) and their cocktail. Differences between the

AN

sensitivities of individual antibodies and their cocktail were found to be statistically significant by group analysis using chi-square test (* indicates P < 0.001 for anti- ESAT-6, $ indicates P <

M

0.01 for anti-Ag85B, and # indicates P < 0.001 for anti-cord factor antibodies for sera samples; € indicates P < 0.001 for anti-ESAT-6, + indicates P < 0.01 for anti-Ag85B and £ indicates P <

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0.001 for anti-cord factor antibodies for pleural/ascitic fluids) (Singh et al., 2016). (d) Recognition of smear-positive (n = 25) and smear-negative (n = 25) PTB patients on the basis of

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antigen detection in sputum and antibody detection in serum (paired sample analysis) by I-PCR. COMB. = Combination of the antigens used; ANY = positive for the presence of any of the 4 RD represents smear-negative cases and

represents smear-positive

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antigen or their antibodies.

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cases (Mehta et al., 2012a).

32

ACCEPTED MANUSCRIPT Table 1. Comparative evaluation of serodiagnostic potential of I-PCR and ELISA based on the detection of mycobacterial antigens and antibodies in body fluids of TB patients. Type of TB

I-PCR % Sensitivity 58.4 (38/65)

EPTB

31.4 (11/35)

Non-TB controls CFP-10

90 (18/20)

PTB

55.4 (36/65)

Respiratory disease controls

ED PT

52.3 (34/65)

CE

Respiratory disease controls EPTB

AC PTB

T

36 (18/50) 85 (17/20)

> 90

Indirect sandwich ELISA: Individual ESAT, CFP-10, CFP-21and MPT64 and cocktail of these antigens were evaluated in TB patients (Kalra et al., 2010a); Indirect sandwich I-PCR: Streptavidin-biotin conjugate was used as a linker for reporter DNA and antibody. Smear microscopy was taken as the reference standard for PTB cases and CRS was taken as the reference standard for EPTB cases (Mehta et al., 2012a).

31.4 (11/35)

Non-TB controls

85 (17/20)

60 (39/65)

Respiratory disease controls

Non TB controls

> 90

90 (18/20)

PTB

EPTB

36 (18/50)

90 (18/20)

25.7 (9/35)

Non-TB controls

MPT-64

> 90

M

EPTB

CFP-21

40 (20/50) 90 (18/20)

Remarks

% Specificity

IP

Respiratory disease controls

% Sensitivity

CR

PTB

% Specificity

US

ESAT-6

ELISA

AN

Antigen/Antibody detection

40 (20/50) 90 (18/20)

> 90

31.4 (11/35) 90 (18/20)

33

ACCEPTED MANUSCRIPT PTB

73.8 (48/65)

Respiratory disease controls EPTB

85 (17/20)

54.3 (19/35)

90 (18/20)

31 (11/35) 90 (18/20)

PTB

83 (151/182)

95 (19/20)

73.6

52.4 (55/105)

Non TB controls

92 (46/50)

56.6 (34/60)

EPTB

PTB

AC

Cord Factor

CE

Non TB controls

PTB

93.3 (28/30)

96.6

50 (30/60) 92.8(39/42)

42.8 (18/42)

Non TB controls Ag85B

97.6 (41/42)

(29/30)

Respiratory disease controls EPTB

50 (30/60)

28.5 (12/42)

60 (36/60)

95.2 (40/42)

Indirect sandwich I-PCR: SMCC was used as a linker for reporter DNA and antibody. CRS was taken as the reference standard. Ag85B detection was found to be superior to the detection of a cocktail of antigens (Ag85B+ESAT6+cord factor) (Mehta et al., 2016).

35.7 (15/42) 93.3 (28/30)

80 (48/60)

Indirect sandwich I-PCR: SMCC was used as a linker for reporter DNA and antibody. CRS was taken as the reference standard for PTB and EPTB cases (Singh et al., 2015).

96 (48/50)

95.2 (40/42)

38 (16/42)

PT

Respiratory disease controls

M

PTB

ED

ESAT-6

86.3 (151/175)

US

68.6 (72/105)

AN

EPTB

90.3 (158/175)

CR

(134/182) Respiratory disease controls

T

Non TB controls Ag85B

54 (27/50)

IP

Cocktail of ESAT-6 + CFP10+CFP-21 + MPT-64

93.3 (28/30) 70 (42/60)

34

ACCEPTED MANUSCRIPT Respiratory disease controls EPTB

92.8 (39/42)

66.6 (28/42)

76.6 (46/60)

Respiratory disease controls

92.8 (39/42)

61.9 (26/42)

Non-TB controls

90 (27/30)

72 (54/75)

Respiratory disease controls

90.5 (38/42)

65.4 (34/52)

92.9 (39/42)

36.5 (19/52) 90 (27/30)

93.3(28/30)

AC

CE

PT

EPTB

93.3 (28/30)

50.7 (38/75)

M

PTB

Non-TB controls

50 (21/42)

ED

PstS1

92.8 (39/42)

US

EPTB

66.6 (40/60)

IP

PTB

93.3 (28/30)

T

90 (27/30)

AN

Cocktail of Ag85B+ESAT6+cord factor

52.3 (22/42)

CR

Non TB controls

95.2 (40/42)

Anti- ESAT-6 antibody

Anti-CFP-10

PTB

40 (41/102)

20.5 (21/102)

Respiratory disease controls

90 (18/20)

Healthy controls

90 (18/20)

PTB

43.1(44/102)

> 90

26.4

Indirect RT-I-PCR: Streptavidin-biotin conjugate was used as a linker for reporter DNA and antibody. CRS was taken as the reference standard. The method could quantify a wide dynamic range of PstS1 (10 ng/mL to 1 pg/mL) in body fluids of TB patients (Sharma et al., 2017). Indirect ELISA: Individual antiESAT-6, anti-CFP10, anti-CFP-21, anti-MPT-64 antibodies and their cocktail were evaluated in PTB patients (Kalra et al, 2010a); Indirect I-PCR:

35

ACCEPTED MANUSCRIPT antibody

(27/102) 95 (19/20)

Healthy controls

95 (19/20)

90 (18/20)

Healthy controls

95 (19/20)

PTB

35.3 (36/102)

> 90

M

95 (19/20)

90 (18/20)

ED

Healthy controls

70.5 (72/102)

PT

PTB

49% (50/102) 85 (17/20)

Healthy controls

85 (17/20)

CE

Respiratory disease controls

AC

Anti-Ag85B antibody

> 90

20.5 (21/102)

Respiratory disease controls

Anti-ESAT6+anti-CFP10+anti-CFP21+anti-MPT-64 antibodies

IP

Respiratory disease controls

T

24.5 (25/102)

US

Anti-MPT-64 antibody

36.2 (37/102)

CR

PTB

> 90

AN

Anti-CFP-21 antibody

Respiratory disease controls

Streptavidin-biotin conjugate was used as linker for reporter DNA and antibody. Smear microscopy was taken as the reference standard for PTB cases and CRS was taken as the reference standard for EPTB cases. Detection of a cocktail of antiESAT-6+anti-CFP10+anti-CFP21+anti-MPT-64 antibodies showed highest sensitivity compared to the detection of individual anti-RD antibodies (Mehta et al., 2012a).

PTB

69.3 (61/88)

97 (73/75)*

60.2 (53/88)

Respiratory disease controls

88.6 (39/44)

90.9 (40/44)

Healthy controls

93.3 (28/30)

96.6 (29/30)

EPTB (Sera)

65 (26/40)

52.5 (21/40)

Indirect I-PCR: SMCC was used as a linker for reporter DNA and antibody. CRS was taken as reference standard for PTB and EPTB cases. Detection of a cocktail of antiAg85B. ESAT-6 and anti-cord factor antibodies showed

36

ACCEPTED MANUSCRIPT Non-TB controls (Sera) 60 (24/40)

63.6 (56/88)

Healthy controls

96.6 (29/30) 57.5 (23/40)

CE

37.5 (15/40)

96 (24/25)

79.5 (70/88)

AC

PTB

96 (24/25)

M 50 (20/40)

PT

Non-TB controls (Pleural/Ascitic fluids)

100 (30/30)

45 (18/40)

92 (23/25)

ED

EPTB (Pleural/Ascitic fluids)

95.4 (42/44)

US

93.1(41/44)

Non-TB controls (Sera)

Anti-cord factor antibody

54.5 (48/88)

Respiratory disease controls

EPTB (Sera)

92 (23/25)

IP

PTB

96 (24/25)

AN

Anti-ESAT-6 antibody

45 (18/40)

96 (24/25)

64.7 (57/88)

Respiratory disease controls

88.6 (39/44)

86.3 (38/44)

Healthy controls

90 (27/30)

86.6 (26/30)

EPTB (Sera)

Non-TB controls (Sera)

highest sensitivity as compared to the detection of individual antibodies (Singh et al., 2016).

T

Non-TB controls (Pleural/Ascitic fluids)

92 (23/25)

CR

EPTB (Pleural/Ascitic fluids)

92 (23/25)

70 (28/40)

52.5 (21/40) 88 (22/25)

92 (23/25)

37

ACCEPTED MANUSCRIPT 67.5 (27/40)

84.1 (73/88)

68.1 (60/88) 90.9 (40/44)

Healthy controls

93.3 (28/30) 77.5 (31/40)

90 (27/30)

65 (26/40)

92 (23/25)

70 (28/40)

Non-TB controls (Pleural/Ascitic fluids)

M

EPTB (Pleural/Ascitic fluids)

ED

Non-TB controls (Sera)

90.9 (40/44)

US

Respiratory disease controls

EPTB (Sera)

92 (23/25)

T

PTB

AN

Anti-Ag85B+ anti-ESAT-6+ anti-cord factor antibodies

92 (23/25)

IP

Non-TB controls (Pleural /Ascitic fluids)

47.5 (19/40)

CR

EPTB (Pleural/Ascitic fluids)

52.5 (21/40)

96 (24/25)

PT

96 (24/25)

92 (23/25)

AC

CE

Respiratory disease controls include patients suffering from respiratory diseases other than TB such as asthma, COPD and non-TB pneumonia; Non-TB controls include controls patients suffering from cancer, trauma, renal failure and non-TB gastrointestinal diseases; Healthy controls were BCG vaccinated with unknown PPD status; * Controls (n = 75) comprised of healthy individuals (n=40), TB contacts (n = 20) and respiratory disease controls (n = 15)

38

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Fig. 1

39

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 2

40

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

M

AN

US

Fig. 3

41

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

Fig. 4

42

ACCEPTED MANUSCRIPT Highlights Immuno-PCR (I-PCR) combines ELISA and PCR.



Antigens/antibodies were detected in TB patients by I-PCR.



I-PCR showed better results than ELISA.



I-PCR assay may facilitate an early diagnosis of TB patients.

AC

CE

PT

ED

M

AN

US

CR

IP

T



43