Combined rapid (TUBEX) test for typhoid-paratyphoid A fever based on strong anti-O12 response: design and critical assessment of sensitivity

Abstract

Rapid diagnostics can be accurate but, often, those based on antibody detection for infectious diseases are unwittingly underrated for various reasons. Herein, we described the development of a combined rapid test for two clinically-indistinguishable bacterial diseases, typhoid and paratyphoid A fever, the latter fast emerging as a global threat. By using monoclonal antibodies (mAbs) to bacterial antigens of known chemical structures as probes, we were able to dissect the antibody response in patients at the level of monosaccharides. Thus, a mAb specific for a common lipopolysaccharide antigen (O12) found in both the causative organisms was employed to semi-quantify the amounts of anti-O12 antibodies present in both types of patients in an epitope-inhibition particle-based (TUBEX) immunoassay. This colorimetric assay detected not only anti-O12 antibodies that were abundantly produced, but also, by steric hindrance, antibodies to an adjoining epitope (O9 or O2 in the typhoid or paratyphoid bacillus, respectively). Sensitivity and, particularly, reaction intensities, were significantly better than those obtained using an anti-O9 or anti-O2 mAb-probe in the examination of paired sera from 22 culture-confirmed typhoid patients (sensitivity, 81.8% vs 75.0%) or single sera from 36 culture-confirmed paratyphoid patients (52.8% vs 28.6), respectively. Importantly, sensitivity was better (97.1% for typhoid, 75.0% for paratyphoid) if allowance was made for the absence of relevant antibodies in certain specimens as determined by an independent, objective assay (ELISA)--such specimens might have been storage-denatured (especially the older paratyphoid samples) or procured from non-responders. Benchmarking against ELISA, which revealed high concordance between the two tests, was useful and more appropriate than comparing with culture methods as traditionally done, since antibody tests and culture target slightly different stages of these diseases. Paired sera analysis was insightful, revealing 64% of typhoid patients who had no change in antibody titer over 4-16 days, and 14% with no IgM-IgG class-switching.

Figure 1. Schematic diagram illustrating the various types of TUBEX tests and the various types of antibodies detected by these.

Shown are the various mAb-indicators (anti-O9, anti-O2 or anti-O12b) and antigenic epitopes (O9, O2 O12a, O12b and O12c) found in S. Typhi or S. Paratyphi A LPS. Arrow indicates antigenic epitope that antibodies from patients presumptively bind to and inhibit binding of the indicator particle.

Figure 2. Pictoral results showing efficacy of various TUBEX tests in detecting mAb-spiked test samples.

Various mAb-indicators (anti-O9, anti-O2 or anti-O12b) in conjunction with O9 LPS- (A) or O2 LPS-coupled (B) magnetic particles to detect various types of purified mAbs (anti-O9, anti-O2 or anti-O12b, or combinations of these). Numericals in microwells denote individual mAb concentration (µg/ml), same for all sets of microwells; ‘score’ denotes TUBEX results.

Figure 3. Pictoral results showing efficacy of various TUBEX tests in detecting clinical samples.

Sera obtained from typhoid (A) or paratyphoid A (B) patients, or from healthy individuals (A), were examined as described in Figure 2. N1-N4, healthy individuals; T1, typhoid individual; T2, pool of 4 typhoid patients; T3, pool of 4 other typhoid patients; PA1-PA6, paratyphoid A patients; numericals in microwells denote TUBEX scores; ND, not done (insufficient specimen).

Figure 4. Comparison of the results scores between TUBEX TF and TUBEX 12T in typhoid patients.

Based on the frequency of the various scores (0–10) (A), or on each individual patient (total 22 patients) (B).

Figure 5. Change in antibody titer (TUBEX score) over time in individual typhoid patients.

TUBEX 12T or TUBEX TF was used to examine the 1^st and 2^nd specimen from 22 patients. Cases divided between those whose 1^st specimens were obtained less than 11 days of fever, and those obtained after 19 days; indicated are ones showing significant rise or fall in antibody titer.

Figure 6. Regression analysis of various ELISA and TUBEX results obtained from typhoid patients.

(Based on Table S1.)