Development of a Prototype Lateral Flow Immunoassay for Rapid Detection of Staphylococcal Protein A in Positive Blood Culture Samples

Abstract

Bloodstream infection (BSI) is a major cause of mortality in hospitalized patients worldwide. Staphylococcus aureus is one of the most common pathogens found in BSI. The conventional workflow is time consuming. Therefore, we developed a lateral flow immunoassay (LFIA) for rapid detection of S. aureus-protein A in positive blood culture samples. A total of 90 clinical isolates including 58 S. aureus and 32 non-S. aureus were spiked in simulated blood samples. The antigens were extracted by a simple boiling method and diluted before being tested using the developed LFIA strips. The results were readable by naked eye within 15 min. The sensitivity of the developed LFIA was 87.9% (51/58) and the specificity was 93.8% (30/32). When bacterial colonies were used in the test, the LFIA provided higher sensitivity and specificity (94.8% and 100%, respectively). The detection limit of the LFIA was 10⁷ CFU/mL. Initial evaluation of the LFIA in 20 positive blood culture bottles from hospitals showed 95% agreement with the routine methods. The LFIA is a rapid, simple and highly sensitive method. No sophisticated equipment is required. It has potential for routine detection particularly in low resource settings, contributing an early diagnosis that facilitates effective treatment and reduces disease progression.

Keywords: Staphylococcus aureus; bloodstream infection; lateral flow immunoassay; positive blood culture samples; rapid detection.

Figure 1

Lateral flow immunoassay protocol for direct detection of Staphylococcus aureus in positive blood culture bottles. +, positive result; -, negative result.

Figure 2

UV–Vis spectra of colloidal gold alone (solid line) and gold–antibody conjugate (dashed line).

Figure 3

Testing of various nitrocellulose membranes (a1 & a2), sample pads (b1 & b2) and running buffers (c1 & c2) for lateral flow immunoassay. (a2–c2) Histogram representing peak area of the test line responding from (a1–c1). 1, Staphylococcus aureus (NCTC10442); 2, Staphylococcus haemolyticus (CNSP40). PC, positive control; NC, negative control. Buffer 1, 50 mM Tris-HCl, 200 mM NaCl and 1% Triton X-100; buffer 2, 50 mM Tris-HCl and 1% Triton X-100; buffer 3, 50 mM Tris-HCl and 1% Tween 20; buffer 4, 5 mM phosphate buffer with a pH of 7.4 and 1% Triton X-100; buffer 5, 5 mM phosphate buffer with a pH of 7.4 and 1% Tween 20. C, control line; T, test line; +, positive result [two red lines of both test and control lines]; -, negative result [one red line of only the control line].

Figure 4

Detection limit of lateral flow immunoassay for identification of protein A in Staphylococcus aureus cells (a1–a3) and purified protein A (b1–b3). (a2 & b2) Calibration curve of lateral flow immunoassay with different concentrations of S. aureus and purified protein A responding from (a1 & b1). (a3 & b3) Histogram representing peak area of the test line responding from (a1 & b1). The red dashed line corresponds to the threshold value (peak area = 100). With 72.52–595.24 nM of purified protein A, the lateral flow immunoassay showed results due to the “hook” effect. C, control line; T, test line; +, positive result [two red lines of both test and control lines]; -, negative result [one red line of only the control line].

Figure 5

Positive and negative results of lateral flow immunoassay for detecting Staphylococcus aureus in bacterial colonies (a) and spiked blood cultures (b). 1, Staphylococcus aureus (NCTC10442); 2, Staphylococcus haemolyticus (CNSP40). C, control line; T, test line; +, positive result [two red lines of both test and control lines]; -, negative result [one red line of only the control line].

Figure 6

Graph plotting of all the results by groups (testing with spiked blood culture samples). The threshold value to determine a positive/negative result is 100 of peak area (red dashed line). The peak area of ≤100 indicated a negative result, whereas that of >100 indicated a positive result.

Figure 7

Stability of the lateral flow immunoassay strip after storage for six months (1–6). (a) The photo images of the lateral flow immunoassay strip. (b) Histogram representing peak area of the test line responding from (a). PC, positive control (10⁷ CFU/mL of Staphylococcus aureus (NCTC10442)); NC, negative control (10⁸ CFU/mL of Staphylococcus haemolyticus (CNSP40)). C, control line; T, test line; +, positive result [two red lines of both test and control lines]; -, negative result [one red line of only the control line].