Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR

Mehta, Promod K; Singh, Netrapal; Dharra, Renu; Dahiya, Bhawna; Sharma, Sunil; Sheoran, Anita; Gupta, K. B.; Chaudhary, Dhruva; Mehta, Neeru; Varma-Basil, Mandira

doi:10.1016/j.mimet.2016.05.003

Cited by 14 publications

(8 citation statements)

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“…However, we could not calculate the recovery of the MB-GNP-I-PCR as it is the only qualitative and affirmative test indicating either the presence or the absence of ESAT-6 marker, but the theoretical recovery appears to be 100% since the LOD of ESAT-6 remained the same (10 fg/mL) in both PBS and spiked saliva samples ( Figure 7A and C). It is intriguing that the LOD of purified ESAT-6 by MB-GNP-I-PCR was ten-fold lesser than the conventional I-PCR performed in robostrip wells 3 (solid format). This could be due to aggregation of NPs in aqueous system resulting from nonspecific binding of functionalized GNPs to the solid phase or the salts present in reagents might be altering the optical, electrical and mechanical properties of NPs.…”

Section: Resultsmentioning

confidence: 99%

“…5,6 In fact, ESAT-6 is an immunodominant T-cell stimulatory antigen recognized by specific interferon-γ secreting T cells, which are present in higher numbers in TB patients with active infection than in uninfected individuals. 7 Strikingly, I-PCR could detect a significantly higher number of samples from smear-negative pulmonary and paucibacillary extrapulmonary TB patients 3,8 and thus showed superiority in sensitivity over the routine diagnostic methods, ie, smear, histopathology and nucleic acid amplification tests such as PCR. Coupling of detection antibodies with the reporter DNA is a crucial step of I-PCR, and various strategies are employed such as streptavidin-protein A, streptavidin-biotin setup, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC) system and recombinant phage particle where surface displayed single-chain variable fragments and phage DNAs themselves act as detection antibodies and DNA tag, respectively, in phage display-mediated I-PCR.…”

Section: Introductionmentioning

confidence: 99%

“…During the last two decades, an unprecedented interest has been emerged for designing new TB diagnostic tools but still a reliable and accurate test for rapid diagnosis for all forms of TB, ie, pulmonary, extrapulmonary, TB-HIV coinfection and latent TB is lacking. We developed immuno-PCR (I-PCR) assay based on the detection of array of Mycobacterium tuberculosis antigens, ie, Ag85B (Antigen 85B, Rv1886c), ESAT-6 (early secreted antigenic target-6), cord factor (trehalose 6,6′-dimycolate) and PstS1 (phosphate-specific transporter lipoprotein, Rv0934), [2][3][4] which certainly revealed better sensitivity than ELISA. ESAT-6 encoded by region of difference (RD1) is M. tuberculosis complex specific and is considered as a potential biomarker for the diagnosis of TB by ELISA and I-PCR, either alone or in combination with other RD1 and RD2 antigens such as CFP-10 (culture filtrate protein-10, Rv3874) and MPT64 (mycobacterial protein from species tuberculosis-64, Rv1980c).…”

Section: Introductionmentioning

confidence: 99%

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Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Singh¹,

Dahiya²,

Radhakrishnan³

et al. 2018

IJN

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PurposeImmuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen.MethodsGNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR.ResultsTransmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6.ConclusionUnlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Singh¹,

Dahiya²,

Radhakrishnan³

et al. 2018

IJN

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“…SICTT with specific antigens, such as a cocktail of ESAT-6/85B 26 and ESAT-6/CFP-10, 8 are used to improve the specificity of reactions. However, these studies still do not offer a solution for commercial use at large farms.…”

Section: Discussionmentioning

confidence: 99%

Diagnostics of tuberculosis and differentiation of nonspecific tuberculin reactions in animals

Basybekov

Bazarbayev

Yespembetov³

et al. 2018

Brazilian Journal of Microbiology

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Tuberculosis is a serious disease of humans and animals, caused by bacteria of the Mycobacterium genus. This leads to complications in the life of the sick person, and subsequently to death. The cattle, who have been diagnosed with this bacterium, are usually sent to the slaughter, with the result that their livestock is reduced. Mycobacteriosis is also a disease, after determining which cattle are most often sent to slaughter. Such a reduction in livestock numbers has a negative effect on the economy. Of the 300 samples from the animals, 25 cultures of atypical bacteria responding to tuberculin were isolated. A series of tests – intravenous tuberculin test, ophthalmic test, palpebral test, “ZhAT” test, showed that most of the tuberculosis changes in cattle were found in regional lymph nodes more often than in internal organs. In healthy for tuberculosis cows, at the age of 4–9 years, seasonal nonspecific sensitivity to tuberculin is observed. Implementation of the developed express method of glutaraldehyde test on farms in healthy tuberculosis will speed up the diagnosis of tuberculosis and mycobacteriosis in animals that reacted to tuberculin and will exclude short-term nonspecific sensitization of their organism to tuberculin. The introduction of this methodology can be used to diagnose and clearly differentiate the diagnoses of “tuberculosis” and “mycobacteriosis” in cattle. This will cure part of the livestock and reduce the amount of slaughter.

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“…I-PCR has been documented to be a rapid, robust, and highly sensitive method for the detection of mycobacterial antigens up to picogram levels from sputum and pleural fluids of TB patients [ 9 , 18 ]. We demonstrated the utility of I-PCR for the diagnosis of pulmonary and extrapulmonary TB, including paucibacillary smear-negative suspected pleural TB patients with good sensitivities (72–83% for pulmonary TB and 62–77% for pleural TB) and specificities (85–93%) based on the detection of array of Mtb antigens, i.e., regions of differences encoded secreted proteins such as ESAT-6 and MPT-64 (Rv1980c), as well as Ag85B (Rv1886c), PstS1 (Rv0934), and cord factor (trehalose 6,6’-dimycolate) in body fluids, which revealed its superiority over ELISA [ 9 – 11 , 18 ]. We also found that I-PCR based on ESAT-6 detection in CSF was more sensitive in comparison to conventional microbiological (smear/culture) and M-PCR tests for the diagnosis of cerebral tuberculomas.…”

Section: Discussionmentioning

confidence: 99%

Role of Immuno-Polymerase Chain Reaction (I-PCR) in Resolving Diagnostic Dilemma Between Tuberculoma and Neurocysticercosis: A Case Report

et al. 2018

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Patient: Female, 17Final Diagnosis: Tuberculous meningitisSymptoms: Headache • vomiting • loss of appetiteMedication: —Clinical Procedure: —Specialty: Infectious DiseasesObjective:Unusual clinical courseBackground:Tuberculoma and neurocysticercosis (NCC) often show similar clinical and neuroimaging features. Differential diagnosis of these 2 diseases is imperative, as tuberculoma is an active infection that requires immediate anti-tubercular therapy (ATT).Case Report:We present the case of a 17-year-old Indian girl with fever, severe headache, and right 6th cranial nerve palsy. Brain magnetic resonance imaging (MRI) showed multiple tiny ring-enhancing lesions in bilateral cerebral parenchyma with mild perilesional edema, which were initially thought to be NCC, but subsequently were diagnosed as brain tuberculomas. Based on clinical findings, mildly increased choline/creatine ratio (1.35) with slight prominent lipid lactate peak and absence of alanine, succinate peak by magnetic resonance spectroscopy (MRS), and the detection of Mycobacterium tuberculosis (Mtb)-specific early-secreted antigenic target-6 (ESAT-6, Rv3875) protein from the cerebrospinal fluid (CSF) by indirect ELISA, as well as indirect immuno-PCR (I-PCR) assay, diagnosis of brain tuberculomas associated with tuberculous meningitis (TBM) was confirmed, which was followed by ATT. The patient responded well and the symptoms resolved.Conclusions:In this case, multiple ring-enhancing lesions of the brain by MRI were diagnosed as tuberculomas associated with TBM by MRS and indirect ELISA/I-PCR method, thus resolving the diagnostic dilemma.

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Diagnosis of tuberculosis based on the detection of a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor by immuno-PCR

Cited by 14 publications

References 10 publications

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Detection of <em>Mycobacterium tuberculosis</em> purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Diagnostics of tuberculosis and differentiation of nonspecific tuberculin reactions in animals

Role of Immuno-Polymerase Chain Reaction (I-PCR) in Resolving Diagnostic Dilemma Between Tuberculoma and Neurocysticercosis: A Case Report

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