Evaluation of a synthetic peptide for the detection of anti-Mycobacterium tuberculosis curli pili IgG antibodies in patients with pulmonary tuberculosis

Abstract

Tuberculosis (TB) remains a serious threat in underdeveloped areas. Mycobacterium tuberculosis curli pili (MTP), a virulence factor, is a potential biomarker for a reliable point of care (POC) test and was evaluated for its ability to react with Immunoglobulin G (IgG) in TB patients. An MTP synthetic peptide in a slot blot assay was used to screen serum/plasma samples (n = 65) in 3 separate cohorts, including 40 TB positive (16 HIV co-infected), 20 TB negative/HIV negative patients and 5 healthy volunteers. Forty samples were true positives (HIV positive, n = 16), 23 true negatives (HIV negative) and 2 false positives (HIV negative). The McNemar test demonstrated a 3.08% accuracy estimate (CI: -2.1% - 3.08%). This confirms that MTP is expressed during infection, including HIV-TB co-infection, is likely to be suitable for the design of a POC test and supports the validation of MTP for TB detection in larger patient populations.

Keywords: Biomarkers; Curli pili; M. tuberculosis; MTB; MTP; Tuberculosis.

Figure 1.

(A) The secondary structure protein analysis of MTP, (i) synthetic peptide used in this study (demarcated with red lines), (ii) synthetic peptide used by another research group for an MTP-specific ELISA (Alteri et al., 2005) and the (B) similarity index of the blastp analysis showing the homology between the MTP amino acid sequences of M. tuberculosis and M. marinum. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2.

(A) Screening of MTP peptide for anti-MTP antibodies from plasma/serum samples of TB positive and TB negative patients using the slot blot method in duplicate in a five-fold dilution series. (B) An example of a complete blot showing all samples testing positive for TB from the blinded cohort. The controls included human serum albumin (HSA) and PBS (-ve controls).

Figure 3.

Slot blot analysis of Cohort N, Cohort T and blinded Cohort A plotting the Intensity Optical Density (IOD) values of each sample per cohort. A diagnostic cut-off intensity value was obtained by calculating the mean plus 2 standard deviations of the negative control samples using Cohort N and Cohort T. (A) 1 in 5 dilution of the serum samples (p = 0.0008, Mann Whitney U test), (B) 1 in 25 dilution of the serum samples (p = 0.0008, Mann Whitney U test), (C) 1 in 125 dilution of the serum samples (p = 0.0008, Mann Whitney U test). The red dots represent the samples in the blinded Cohort A that fell below the cut off line and belong to the 5 healthy controls (3A and 3B). In the 1 in 125 dilution (3C), the additional 3 red dots below the cut-off line (Cohort N) represents TB false negative samples, indicating that the increased dilution of samples reduced the accuracy of the slot blot assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)