Detection of MPT-64 protein in pleural tuberculosis cases by magnetic bead-gold nanoparticle-PCR amplified immunoassay

ABSTRACT

Aim

Diagnosis of pleural tuberculosis (TB) is challenging; thus, an efficient method is urgently needed.

Methods

We developed a magnetic-bead-gold nanoparticle-PCR amplified immunoassay (MB-AuNP-I-PCR, liquid system) to detect the Mycobacterium tuberculosis MPT-64 protein in pleural TB patients. AuNPs functionalized with detection antibodies/oligonucleotides were characterized by UV-vis spectroscopy, Transmission/Scanning electron microscopy, Fourier-transform infrared spectrometer, ELISA, and PCR, whereas MBs conjugated with detection antibodies were validated by magneto-ELISA/UV-vis spectroscopy.

Results

We utilized the MB-AuNP-I-PCR for MPT-64 detection in 99 clinical specimens which displayed 85.2% sensitivity and 97.8% specificity to diagnose pleural TB cases. Markedly, the sensitivity achieved by MB-AuNP-I-PCR was noticeably higher (p < 0.01) than magneto-ELISA and GeneXpert.

Conclusion

This is a preliminary report to diagnose pleural TB cases by MB-AuNP-I-PCR with promising results that require further corroboration in a higher number of specimens.

Plain Language Summary

Pleural tuberculosis is a sickness that is hard for doctors to diagnose because it doesn’t show clear signs. The current tests that doctors use don’t work very well, so we designed a new test using nanoparticles. This test showed promise in comparison to the other tests, but our findings need to be confirmed with a larger sample size.

1. Introduction

Extrapulmonary tuberculosis (TB) constitutes ~15-20% of the TB burden, wherein pleural TB/TB pleuritis or TB pleural effusion is the second most common manifestation (~25%) after TB lymphadenitis [1]. Pleural TB diagnosis poses challenges largely through atypical clinical presentations and scanty bacillary content in specimens [2]. The timely/dependable diagnosis is pivotal to initiating anti-tubercular therapy (ATT). In developing nations, smear microscopy is commonly employed to diagnose pleural TB, but it has low sensitivity [3]. Although the culture of pleural fluids/biopsies is the reference standard, it often provides low sensitivity on Lowenstein-Jensen (LJ) medium and has a delayed turnaround time of 4–6 weeks [4]. Comparatively, the culture on MGIT-960 has a shorter turnaround time of ~12-14 days, but this is not available in several reference laboratories of developing nations [3]. Though GeneXpert and GeneXpert Ultra are the most important advancements in TB diagnosis, their high cost is the main obstruction, especially in resource-limited settings [5].

Formerly, anti-Plc1 (Rv2351c)+anti-HspX (Rv2031c)+anti-ESAT-6 (Rv3875)+anti-TB 8.4 (Rv1174c) IgG antibodies were identified by ELISA in pleural TB patients with good diagnostic yield [6]. While detecting anti-cord factor+anti-ESAT-6+anti-Ag85B (Rv1886c) antibodies by immuno-PCR (I-PCR) in several body fluids of extrapulmonary TB cases including pleural fluids, Singh et al. [7] demonstrated a moderately good sensitivity/specificity. The WHO [8] disapproved of the utility of antibody identification owing to incorrect results, while detection of Mycobacterium tuberculosis (Mtb) antigen is more accurate as it directly detects the active disease. We previously detected Ag85B in extrapulmonary TB specimens by I-PCR with a moderately good sensitivity/specificity [9]. Noticeably, I-PCR combines ELISA/PCR to detect oligonucleotides, the surrogate biomarkers for protein/lipid antigens [10]. Various mycobacterial antigens, i.e., MPT-64 (Rv1980c), CFP-10 (Rv3874), ESAT-6, lipoarabinomannan (LAM), etc. were identified by I-PCR to diagnose TB cases [10,11]. Coupling of oligonucleotides/detection antibodies is vital for I-PCR that mostly exploits streptavidin-biotin or covalent coupling via carboxylate salt but that is tedious [12]. Comparatively, conjugation of gold nanoparticles (AuNPs) with oligonucleotides/detection antibodies is straightforward, and those functionalized AuNPs may enhance the sensitivity of the assay [12]. To further improve the innovation and diagnostic performance of I-PCR, this study was planned to detect the Mtb biomarkers in pleural TB specimens by magnetic bead-AuNP-PCR amplified assay (MB-AuNP-I-PCR), where functionalized AuNPs are attached with MB-capture antibody-antigen complexes [13]. Initially, Perez et al. [14] devised this assay (named “NP amplified I-PCR”) for the detection of respiratory syncytial virus surface protein using antibody-functionalized MBs and Synagis antibody conjugated with AuNP co-functionalized with thiolated DNA, which exhibited several-fold enhanced sensitivity in the detection limit, as compared to ELISA and reverse transcription-PCR. We also detected the Mtb MPT-64+CFP-10/MPT-64+LAM biomarkers in clinical specimens of pulmonary/extrapulmonary TB cases by MB-AuNP-I-PCR [15,16], although exclusive pleural TB cases were not exploited by this assay.

Among the mycobacterial Ag85 complex, Ag85B is the main protein (~41%) of total culture supernatant, which is an attractive biomarker to diagnose active TB by I-PCR/ELISA [9]. Meanwhile, MPT-64 (24-kDa), the Mtb-specific biomarker of active disease, constitutes ~8% of culture filtrate protein that is absent in non-tuberculous mycobacteria and most of Mycobacterium bovis BCG substrains [16]. Moreover, the Standard Diagnostics Bioline TB kit for MPT-64 detection confirmed the pulmonary TB specimens in children [17]. We first made a comparative evaluation to detect the individual Ag85B and MPT-64 proteins in pleural TB patients by MB-AuNP-I-PCR, in a pilot study (n = 45). After establishing the superiority of MPT-64 over Ag85B for diagnosing pleural TB, we focused on MPT-64 detection in clinical specimens (n = 99) of pleural TB cases/non-TB controls by MB-AuNP-I-PCR vs. magneto-ELISA, albeit some specimens were subjected to GeneXpert.

2. Methods

2.1. Reagents

Purified MPT-64, Ag85B, and anti-Mtb CDC1551 polyclonal antibodies (pAbs) were obtained from BEI Resources, Manassas, USA, whereas rabbit anti-MPT-64/Ag85B pAbs were purchased from Abcam, Cambridge, UK.

2.2. Clinical samples

Pleural biopsies (n = 23) and pleural fluids (n = 65) were obtained from the suspected pleural TB patients/non-TB controls attending the OPD of UHS, Rohtak, while pus specimens (n = 11) were obtained from the suspected TB empyema patients, a rare form of pleural TB [3,18]. Patients provided written informed consent, and this work was approved by the “Human Ethics Committee” (HEC/2023/46), MDU, Rohtak. Initially, this study had 126 patients with symptoms like nonproductive cough, low-grade fever, chest pain, dyspnea, weight loss, etc [4]. Based on exclusion criteria (Figure 1(a)), 27 patients were excluded, and 99 patients were recruited, which were grouped as per composite reference standard (CRS) criteria [19]. The CRS included several tests, viz. smear/culture, imaging, histopathological examination (HPE)/cytology, PCR, adenosine deaminase (ADA, ≥40 IU/L) activity, and ATT response [19]. Any patient positive for ≥1 CRS criteria was regarded as “strongly suspected pleural TB” (n = 54) though the individuals who were non-TB and had different diagnoses were categorized as non-TB controls (n = 45). The strongly suspected cases were further classified as (i) confirmed cases (n = 5) in terms of positive-smear microscopy/culture on LJ or HPE, (ii) clinically suspected cases (n = 49) in terms of clinical findings, PCR, cytology, imaging, ATT response, etc [20]. The non-TB controls (n = 45) included pleural biopsies (n = 9) and pleural fluids (n = 36) of cancer, renal failure, and trauma patients.

Figure 1.

(a) The flowchart for grouping of the study participants (strongly suspected pleural TB cases/non-TB controls) for MPT-64 detection by MB-AuNP-I-PCR vs. magneto-ELISA. (b) The flowchart for grouping of the study participants (clinically suspected pleural TB cases/non-TB controls) by GeneXpert vs. MB-AuNP-I-PCR.

We evaluated the efficacy of MB-AuNP-I-PCR vs. magneto-ELISA for MPT-64 detection in 99 clinical specimens; the categorization of the same has been shown (Figure 1(a)). Moreover, 42 pleural samples (16 pleural biopsies and 26 pleural fluids) were randomly chosen for GeneXpert vs. MB-AuNP-I-PCR (Figure 1(b)).

2.3. Processing of samples

Clinical samples were decontaminated/processed in the biosafety cabinet using 1% N-acetyl L-cysteine +4% NaOH [21], while pleural fluids were directly centrifuged, and the supernatants were stored at −20°C after filter-sterilization.

2.4. AuNPs synthesis and their characterization

The synthesis of ~20 nm AuNPs and their morphology (size/shape) was investigated by Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) as described previously [13]. The mean particle size was determined by ImageJ software, whereas High-resolution (HR) TEM was utilized to ascertain the planar lattice (d) spacing [22]. The morphology of commercial MBs was observed by SEM, while energy-dispersive X-ray spectroscopy (EDX) was utilized to confirm the elemental composition of AuNPs [13]. AuNP solution had 8.8 × 10⁹ particles/mL, as found by Nanosight NS300 NTA (nanoparticle tracking analysis).

2.5. Functionalization of AuNPs with detection antibodies (anti-MPT-64 pAbs)/oligonucleotides

To functionalize AuNPs with detection antibodies/oligonucleotides, 500 μL of each of AuNPs and detection antibodies (1:500) were incubated for 45 min at room temperature (RT) on a dancing shaker [23]. The rest of the steps were mentioned in a previous paper [15]. Functionalized AuNPs were dissolved in phosphate-buffered saline (PBS) and had 1.6 × 10⁹ particles/mL, which were characterized by UV-vis spectroscopy and TEM/HRTEM. Further, the detection antibodies/signal DNA in functionalized AuNPs was investigated by indirect ELISA/conventional PCR [15,24]. The presence of specific functional groups in AuNPs, AuNPs+detection antibodies, and functionalized AuNPs was observed by the Varian 7000 FTIR (Fourier-transform infrared) spectrometer.

2.6. Coupling of MBs with capture antibodies

MBs conjugated with capture antibodies (anti-Mtb pAbs) were dissolved in 500 μL of PBS [15,23] and stored at 4°C. Validation of the same was carried out with magneto-ELISA and UV-vis spectroscopy by measuring ODs at 405 nm and 270 nm, respectively [15].

2.7. MPT-64/Ag85B detection in clinical specimens by MB-AuNP-I-PCR/magneto-ELISA

For MB-AuNP-I-PCR, 50 µL of “MBs+capture antibodies” (1:1000) were placed in the Eppendorf tubes, followed by washing with “washing buffer” (PBS +0.05% Tween-20) using an external magnetic field, the addition of a blocker (5% BSA solution) and incubation at RT for 2 hr [15]. After washing, 50 µL of various diluents of purified MPT-64/Ag85B (1 µg/mL-10 µg/mL in PBS) or optimally diluted clinical samples were added and incubated at RT for 1 hr. The rest of the steps was the same as explained previously [15]. All the clinical specimens were run in duplicates to ensure their reproducibility. A specific amplicon on 4% gel (76 bp) implies antigen identification by MB-AuNP-I-PCR [24].

Likewise, MPT-64 was detected in clinical samples by magneto-ELISA in the Eppendorf tubes as detailed by Dahiya et al. [15] with slight modifications, where instead of functionalized AuNPs, anti-MPT-64 pAbs (1:500), and goat anti-rabbit IgG alkaline phosphatase (1:1000) were employed. The OD was measured at 405 nm in the ELISA reader. To investigate the sample matrix effect, the pleural fluids (n = 5) of non-TB individuals were spiked with purified antigen (1 μg/mL), and the limit of detection (LOD) was noted by MB-AuNP-I-PCR/magneto-ELISA [15,24].

2.8. GeneXpert assay

Only some of the pleural biopsies/fluids from 42 individuals (21 clinically suspected pleural TB and 21 non-TB controls) underwent GeneXpert owing to its high cost, wherein 1 mL of pleural fluid/biopsy homogenate was mixed with 2 mL of bacterial buffer and incubated at RT for 15 min [21]. Two mL of this mixture was added to the Xpert cartridge and later inserted into the instrument. The assay was carried out at the Microbiology Department, UHS, Rohtak, and those results were directly compared with MB-AuNP-I-PCR.

2.9. Statistical analysis

For magneto-ELISA, the mean OD of clinical samples from the control group + 2SD was taken as the cutoff value for a positive sample [16]. Comparisons of ODs between different groups were made using the 2-sample t-test with equal variances, while sensitivity/specificity values of MB-AuNP-I-PCR, magneto-ELISA, and GeneXpert were calculated at 95% confidence interval (CI), against CRS [15,16]. Comparisons of MB-AuNP-I-PCR vs. magneto-ELISA/GeneXpert were made against CRS, using a chi-square (X²) test [15,16]. The statistical software SPSS version 20 was utilized for the analysis.

3. Results

In a pilot study, 19 out of 23 clinically suspected pleural TB cases were shown to be positive for both MPT-64 and Ag85B identification by MB-AuNP-I-PCR, demonstrating a sensitivity of 82.6%, against CRS. Among 22 non-TB controls, 1 and 3 subjects exhibited false-positive results for MPT-64 and Ag85B detection by MB-AuNP-I-PCR, yielding specificities of 95.5% and 86.4%, respectively. Since the MPT-64 detection exhibited somewhat higher specificity than Ag85B detection by MB-AuNP-I-PCR and equally good sensitivity like Ag85B detection, we chose MPT-64 detection by MB-AuNP-I-PCR in a relatively larger number of specimens (n = 99) and those results were compared with magneto-ELISA.

The UV-vis spectroscopy of ~20 nm AuNPs showed the presence of a single peak at 520.6 nm (Supplementary Figure S1(a)). The synthesized AuNPs revealed the presence of stable, spherical, and well-dispersed particles by TEM (Figure 2(a(i))), wherein the average size of AuNPs was measured using ImageJ software. The size histogram showed that the average size of AuNPs was 17.67 ± 1.25 nm (ranging from 16–20 nm) (Figure 2(a(ii))). The SEM images also revealed the spherical morphology of AuNPs with smooth surfaces (Supplementary Figure S1(b)), whereas irregular shapes (i.e., diamond, brick, rectangular, etc.) were noted for commercial MBs (Supplementary Figure S1(d)). The EDX spectrum showed a characteristic peak at 2.12 keV for the elemental Au of AuNPs (Supplementary Figure S1(c)).

Figure 2.

Physicochemical characterization of AuNPs, AuNPs+detection antibodies and functionalized AuNPs by TEM: (a) AuNPs (i) TEM image at 200 KX magnification revealed spherical monodispersed AuNPs (ii) size histogram revealed an average size of AuNPs ranging between 16 to 20 nm and (iii) HRTEM at 800 KX magnification revealed the crystal lattice arrangement of AuNPs. (b) AuNPs+detection antibodies (i) image at 200 KX (ii) size histogram for TEM with an average size of 15–22 nm and (iii) HRTEM of AuNPs+detection antibodies at 800 KX. (c) functionalized AuNPs (i) image at 200 KX (ii) size histogram for TEM with an average size of 16–23 nm and (iii) HRTEM of functionalized AuNPs at 800 KX.

Furthermore, the UV-vis spectroscopy exhibited a shift in the absorbance maxima peak of AuNPs from 520.6 to 528.2 nm, when functionalized with detection antibodies/oligonucleotides (Supplementary Figure S2(a)). The conjugation of detection antibodies to AuNPs was also corroborated by ELISA (Supplementary Figure S2(b)), wherein functionalized AuNPs demonstrated a substantially greater mean OD (p < 0.01), compared with unbound AuNPs. The attachment of signal DNA to functionalized AuNPs was confirmed by conventional PCR, where a specific amplicon (76 bp) was seen (Supplementary Figure S2(c)). TEM micrograph revealed an average size of 18.44 ± 2.4 nm (ranging from 15–22 nm) for AuNPs+detection antibodies and 18.96 ± 2.13 nm (ranging from 16–23 nm) for functionalized AuNPs (Figure 2(b, c(i, ii))). Moreover, HRTEM exhibited the crystal lattice arrangement for AuNPs, AuNPs+detection antibodies, and functionalized AuNPs (Figure 2(a-c(iii))), indicating d spacing of 0.180 nm, 0.161 nm, and 0.116 nm, respectively, between the atomic planes that are consistent with the typically observed lattice spacing d111 for the diffraction plane (111) of AuNPs.

The FTIR spectra demonstrated a broad absorption band between 3200 and 3600 cm⁻¹ in AuNPs, AuNPs+detection antibodies (anti-MPT-64 pAbs), and functionalized AuNPs (Figure 3), which was attributed to O-H and N-H stretching vibration. The characteristic peaks observed in AuNPs+detection antibodies at 2932 cm⁻¹ and 2859 cm⁻¹ and functionalized AuNPs at 2929 cm⁻¹ and 2879 cm⁻¹ corresponded to C-H stretching vibration, while these peaks were not present in the unbound AuNPs, which confirmed the formation of new bonds after functionalization. The observed intense peak at 1640 cm⁻¹ was due to asymmetric and symmetric stretching vibration of C=C or N-H bending in AuNPs, AuNPs+detection antibodies, and functionalized AuNPs. To add, many characteristic peaks were prominent in AuNPs+detection antibodies and functionalized AuNPs between 1000 and 1500 cm⁻¹. Also, the weak peak appearing only in functionalized AuNPs at 997 cm⁻¹ for C=C bonding was not seen in AuNPs and AuNPs+detection antibodies, thus authenticating the functionalization of AuNPs.

Figure 3.

FTIR spectra of AuNPs (red colored), AuNPs+anti-MPT-64 pAbs (detection antibodies) (green colored) and functionalized AuNPs (black colored).

The attachment of capture antibodies with MBs was confirmed by magneto-ELISA, which documented a significantly greater OD (p < 0.01) than unbound MBs (Supplementary Figure S2(d)). This was further corroborated by UV-vis spectroscopy that showed a considerably higher OD (p < 0.01) with MBs+capture antibodies as compared to unbound MBs (Supplementary Figure S2(e)).

Next, the LODs for purified MPT-64 (in PBS) were determined as 1 fg/mL and 500 pg/mL by MB-AuNP-I-PCR (Figure 4) and magneto-ELISA (data not shown), respectively. While spiking pleural fluids of non-TB individuals with purified MPT-64, detection limits of 5 fg/mL and 5 ng/mL were observed by MB-AuNP-I-PCR (Supplementary Figure S3(a)) and magneto-ELISA (data not shown), respectively, showing sample matrix effect by both the techniques.

Figure 4.

LOD for the purified MPT-64 by MB-AuNP-I-PCR, wherein a 76 bp amplicon indicated a positive result on 4% agarose gel: lane M, 20 bp ladder; lanes 1–12, serial ten-fold dilutions of the purified MPT-64 (from 1 μg/mL to 10 ag/mL); lane 13, I-PCR-negative control (no antigen coated, rest all the reagents added); lane 14, PCR-negative control (no template DNA, rest all the reagents added); lane 15, PCR positive control (signal DNA, 1 ng/mL). The experiments were performed twice and one of the representative figures has been shown.

With clinical specimens, we attained sensitivities of 83.7% (95% CI, 70.3–92.7%) and 85.2% (95% CI, 72.9–93.4%) by MB-AuNP-I-PCR in clinically suspected (n = 49) and total pleural TB (n = 54) patients, respectively, against CRS (Table 1). Furthermore, MB-AuNP-I-PCR displayed a considerably larger sensitivity (p < 0.01) than magneto-ELISA in both clinically suspected/total pleural TB patients. Strikingly, a κ-value of 0.8 (p < 0.01) observed for MB-AuNP-I-PCR vs. magneto-ELISA in total pleural TB cases exhibited a substantial agreement. Amid the individual pleural TB specimens, sensitivities were in the descending order as pleural biopsies [92.9% (95% CI, 66.1–99.8%)] >pus [90.9% (95% CI, 58.7–99.8%)] >pleural fluids [79.3% (95% CI, 60.3–92%)] by MB-AuNP-I-PCR (Table 2). Meanwhile, one and four individuals out of 45 non-TB controls showed false-positive results by MB-AuNP-I-PCR and magneto-ELISA, yielding specificities of 97.8% (95% CI, 88.2–99.9%) and 91.1% (95% CI, 78.8–97.5%), respectively (Table 1). Representative examples of positive pleural TB specimens/non-TB controls by MB-AuNP-I-PCR are depicted in Supplementary Figure S3(b).

Table 1.

Sensitivity/Specificity of MB-AuNP-I-PCR and magneto-ELISA for MPT-64 detection at 95% CI in pleural TB cases and non-TB controls, against CRS.

Category of pleural TB	MB-AuNP-I-PCR			Magneto-ELISA
	(+)	% Sensitivity	% Specificity	(+)	% Sensitivity	% Specificity
Confirmed (n = 5)	5	100 (47.8-100)		4	80 (28.4-99.5)
Clinically suspected (n = 49)	41	*83.7 (70.3-92.7)		31	†63.3 (48.3-76.6)
Total (n = 54)	46	*85.2 (72.9-93.4)		35	†64.8 (50.6-77.3)
Non-TB controls (n = 45)	1		97.8 (88.2-99.9)	4		91.1 (78.8-97.5)

*Significant differences (p < 0.01) were observed between the sensitivities of clinically suspected and total pleural TB cases by MB-AuNP-I-PCR vs. magneto-ELISA, using X² test.

Table 2.

Sensitivities obtained in individual pleural TB specimens at 95% CI by MB-AuNP-I-PCR and magneto-ELISA based on MPT-64 detection, against CRS.

Assay	Specimen types	Number of specimens analyzed	Number of positive specimens	% Sensitivity
MB-AuNP-I-PCR	Pleural biopsies	14	13	92.9 (66.1-99.8)
	Pus	11	10	90.9 (58.7-99.8)
	Pleural fluids	29	23	79.3 (60.3-92)
Magneto-ELISA	Pleural biopsies	14	10	71.4 (41.9-91.6)
	Pus	11	7	63.6 (30.8-89.1)
	Pleural fluids	29	18	62.1 (42.3-79.3)

While evaluating 21 pleural specimens of clinically suspected pleural TB cases by GeneXpert, a sensitivity of 23.8% (95% CI, 8.2–47.2%) was obtained (Table 3), which was noticeably lower (p < 0.01) than MB-AuNP-I-PCR [(85.7% (95% CI, 63.7–96.9%)]. However, among 21 non-TB controls, no individual showed false positivity by Xpert, demonstrating a specificity of 100% (95% CI, 83.9–100%).

Table 3.

Sensitivity/Specificity of GeneXpert vs. MB-AuNP-I-PCR for MPT-64 detection at 95% CI in clinically suspected pleural TB cases and non-TB controls, against CRS.

Category of pleural TB	GeneXpert			MB-AuNP-I-PCR
	(+)	% Sensitivity	% Specificity	(+)	% Sensitivity	% Specificity
Clinically suspected (n = 21)	5	*23.8 (8.2-47.2)		18	†85.7 (63.7-96.9)
Non-TB controls (n = 21)	0		100 (83.9-100)	1		95.2 (76.2-99.9)

*Significant difference (p < 0.01) was observed between the sensitivities of MB-AuNP-I-PCR vs. GeneXpert, using X² test.

4. Discussion

In this preliminary study, we utilized the MB-AuNP-I-PCR assay to detect the MPT-64 protein in clinical specimens of pleural TB cases. The synthesized AuNPs (~20 nm) revealed spherical and well-dispersed particles by TEM (Figure 2(a,i)). Markedly, the characteristic peak for elemental Au by EDX confirmed the synthesis of AuNPs (Supplementary Figure S1(c)). Functionalization of AuNPs with detection antibodies/oligonucleotides exhibited a shift in the absorbance maxima peak from 520.6 to 528.2 nm by UV-vis spectroscopy (Supplementary Figure S2(a)). Further, attachment of detection antibodies and signal DNA (76 bp) in the functionalized AuNPs was corroborated by ELISA and PCR, respectively (Supplementary Figure S2(b,c)). These findings agree with the earlier reports to characterize the functionalized AuNPs by TEM/SEM, EDX spectrum, UV-vis spectroscopy, ELISA, and PCR [13,15]. Interestingly, the d spacing between the atomic planes of AuNPs/functionalized AuNPs by HRTEM (Figure 2(a-c), iii) is also in concurrence with a previous report [22], where HRTEM revealed a fine-line spacing of the face-centered cubic structure of AuNPs with a width of 0.28 nm.

The FTIR spectra documented a broad absorption band between 3200 and 3600 cm⁻¹ in AuNPs, AuNPs+detection antibodies, and functionalized AuNPs that was ascribed to O-H and N-H stretching vibration (Figure 3), implying the stabilization of AuNPs through intermolecular hydrogen bonding. Similarly, Borse and Konwar [22] exhibited two main peaks for O-H and C=O stretching at 3307 cm⁻¹ and 1635 cm⁻¹, respectively, for ~30 nm AuNPs, though a medium peak was seen at 1365 cm⁻¹ (related to C-O stretching vibration). The characteristic peaks observed in AuNPs+detection antibodies at 2932 cm⁻¹ and 2859 cm⁻¹ and functionalized AuNPs at 2929 cm⁻¹ and 2879 cm⁻¹ corresponded to C-H stretching vibration, while these peaks were not present in unbound AuNPs, which confirmed the formation of new bonds after functionalization. Likewise, the characteristic peak seen with AuNPs at 3435 cm⁻¹ (attributed to O-H stretching vibrations) was absent in AuNPs conjugated with anti-hepatitis B surface monoclonal antibodies [25].

The LODs for purified MPT-64 were obtained as 1 fg/mL and 500 pg/mL by MB-AuNP-I-PCR and magneto-ELISA, respectively. However, the sample matrix effect was displayed when pleural fluids of non-TB individuals were spiked with purified MPT-64, since higher detection limits of 5 fg/mL and 5 ng/mL were noted for both MB-AuNP-I-PCR and magneto-ELISA, respectively, albeit such effect was relatively less marked for MB-AuNP-I-PCR than the latter (5-fold vs. 10-fold). Our LOD results with purified MPT-64 are almost identical with purified ESAT-6/CFP-10+MPT-64 detection by MB-AuNP-I-PCR [13,15]. Concomitantly, the detection limits of 2 pg/mL and 8 pg/mL were attained for the Plasmodium falciparum histidine-rich protein 2 and COVID-19 nucleocapsid protein, respectively, within human serum by magneto-ELISA [26].

Notably, we acquired a sensitivity of 85.2% and 97.8% specificity by MB-AuNP-I-PCR in 99 cases (54 pleural TB and 45 non-TB controls), against CRS (Table 1), while magneto-ELISA displayed a substantially lower sensitivity (p < 0.01) than MB-AuNP-I-PCR. While comparing MPT-64 identification by MB-AuNP-I-PCR in pleural TB cases of this study with MPT-64+LAM identification within urinary extracellular vesicles (EVs) of genitourinary TB cases by MB-AuNP-I-PCR [16], MPT-64 detection revealed almost similar specificity (97.8 vs. 97.1%) but somewhat lower sensitivity (85.2 vs. 87.2%), compared with MPT-64+LAM identification. This could be due to the detection of two immunodominant antigens and their enhanced concentration within the urinary EVs [16], as compared to MPT-64 detection in neat pleural TB specimens. However, when MB-AuNP-I-PCR results for MPT-64 recognition in exclusive pleural TB specimens of this study were compared with MPT-64+CFP-10 recognition in the total extrapulmonary TB specimens (lymph node aspirates, ascitic/pleural fluids, etc.) by MB-AuNP-I-PCR [15], MPT-64 recognition revealed almost similar specificity (97.8 vs. 97.9%) but higher sensitivity (85.2 vs. 78.1%) than the latter. This was perhaps owing to the different nature of extrapulmonary TB specimens analyzed in the two studies. Moreover, while assessing the individual specimens of strongly suspected pleural TB cases by MB-AuNP-I-PCR, the highest sensitivity was achieved with pleural biopsies (92.9%), followed by pus (90.9%) and pleural fluids (79.3%) (Table 2). The difference in the sensitivities of different pleural specimens could be attributed to dissimilar bacterial load, resulting in different antigen concentrations [3].

GeneXpert demonstrated a significantly lower sensitivity of 23.8% (p < 0.01), compared with MB-AuNP-I-PCR (85.7%) in 21 clinically suspected TB pleurisy cases, against CRS (Table 3), though 100% specificity was achieved in the non-TB control group (n = 21). However, these data agree with the other reports on TB pleurisy diagnosis by Xpert [20,27,28], where low sensitivities (14–29%) and high specificities (98–100%) were documented, against CRS/MGIT-960 culture or HPE. Whilst our MB-AuNP-I-PCR results show promise compared to both Xpert and magneto-ELISA to diagnose pleural TB cases, these findings need to be validated by further investigation. The enhanced sensitivity of MB-AuNP-I-PCR could be due to twofold amplification, since AuNPs liberate multiple signal DNA/antibody and PCR relays an additional level of amplification [14,23]. Whereas high specificity is most likely due to the detection of Mtb-specific MPT-64 and the utility of the MB-AuNP-I-PCR technique associated with vigorous washing/removal of unattached antigens/antibodies, which resulted in the elimination of background noise and yielded low false positivity [15].

The main limitation of this study is that the sample size was relatively small, and our MB-AuNP-I-PCR results need to be authenticated in a higher number of clinical specimens collected from different tertiary hospitals. Further, we did not validate our MB-AuNP-I-PCR results with culture, since only four samples out of 54 strongly suspected pleural TB cases were positive for culture, which were all positive for MB-AuNP-I-PCR, leading to 100% sensitivity. The rest of the 50 strongly suspected pleural TB individuals were culture-negative; thus, MB-AuNP-I-PCR vs. magneto-ELISA results were assessed with CRS parameters, in collaboration with index TB guidelines [19]. However, while comparing our data with clinical findings and PCR (mpt64/IS6110), 22 of 27 strongly suspected pleural TB individuals were positive for MB-AuNP-I-PCR, resulting in a comparable high sensitivity (81.4%) as that achieved using CRS, yet magneto-ELISA (63%) and Xpert (25%) showed lower sensitivities.

5. Conclusion

We designed an MB-AuNP-I-PCR for MPT-64 identification in clinical specimens of pleural TB cases, which revealed high sensitivity/specificity. Moreover, the sensitivity achieved by MB-AuNP-I-PCR was greater than magneto-ELISA and Xpert. This is a preliminary report on MPT-64 detection in pleural TB cases by MB-AuNP-I-PCR with encouraging results, which necessitate further validation in a larger number of specimens obtained from varied geographical locations.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Funding Statement

This work was financially supported by the Indian Council of Medical Research, New Delhi [5/8/25/ITRC/Diag/2022/ECD-1] sanctioned to PK Mehta. A Soni acknowledges the University Grants Commission [Ref. No 221610106016] and DCRUST, Murthal for awarding the Junior Research Fellowship and University Research Scholarship, respectively.

Article highlights

• The diagnosis of pleural TB is difficult and is mainly ascribed to atypical clinical presentations and the paucibacillary nature of clinical specimens.

• The detection limit for the purified MPT-64 was found to be 1 fg/mL by magnetic-bead-gold nanoparticle-PCR amplified immunoassay (MB-AuNP-I-PCR), which was 2 × 10⁵-fold less than magneto-ELISA.

• We utilized the MB-AuNP-I-PCR assay for MPT-64 detection in clinical specimens (n = 99) of pleural TB and non-TB controls and the results were compared with magneto-ELISA.

• We achieved high sensitivity (85.2%) and specificity (97.8%) in 54 pleural TB individuals and 45 non-TB controls, respectively. The sensitivity acquired by MB-AuNP-I-PCR was noticeably superior (p < 0.01) to both magneto-ELISA and GeneXpert, but these findings are more exploratory.

• This is a preliminary study to diagnose pleural TB cases by MB-AuNP-I-PCR that warrants further authentication with a larger sample size.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical declaration

This work was approved by the Human Ethics Committee (HEC/2023/46), MDU, Rohtak.

Authors contributions

A Soni, PK Mehta, and B Dahiya carried out majority of the experiments, participated in data analysis and preparation of the manuscript; A Rais, A Soni, B Dahiya, A Gahlaut, K Nehra, V Raj, and T Prasad carried out some experiments; T Prasad, A Rais, B Dahiya, R Sheoran, and PK Mehta participated in data analysis; PK Mehta conceived the study and designed the experiments; PK Mehta, A Soni, and B Dahiya prepared the final draft of the manuscript. All the authors edited the manuscript and approved the final version of the manuscript.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17460913.2024.2432179