Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies

Abstract

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Keywords: LFA; Valley fever; coccidioidomycosis; diagnostic.

Fig 1

(A) Schematic of anti-CTS1 antibody LFA. Patient serum or plasma is added to the sample pad and chased with buffer to promote lateral flow. If anti-CTS1 antibodies are present, a mixture of antibody complexes to CTS1 and CTS1 coupled to gold nanoparticles (GNP) will be formed, creating a visible test line. If no anti-CTS1 antibodies are present, no complexes will be formed, and no test line will be observed. (B) Examples of LFA results for four patients. The red control line adjacent to the “C” on the cassette indicates the test ran properly, while the presence or absence of a red line adjacent to the “T” on the cassette is used to measure the level of anti-CTS1 antibodies in the sample tested. (1) represents a patient with a negative result with test line density units of 17,470; (2) represents patient serum with a test line density of 93,266 and CF titer of 1:2; (3) represents patient serum with a test line density of 371,565 and CF titer of 1:32; and (4) represents patient serum with a test line density of 5,56,583 and CF titer of 1:256. CF titers are those reported by the reference laboratory (Mayo Clinic Laboratories).

Fig 2

Prevalence of anti-CTS1 antibodies in human sera. The group on the left of the graph includes patients who were positive by CF and/or ID: samples that were positive by both CF and ID are represented by black circles; samples positive by CF but negative by ID (IgG and/or IgM) are shown as grey squares; and samples negative by CF but positive by ID (IgG and/or IgM) are shown as blue diamonds. The group in the middle of the graph includes patients who were negative by CF and ID: samples that were positive by EIA IgG are shown as green triangles and samples negative by EIA are shown as black inverted triangles. The group on the right of the graph shows patients with positive serology to other endemic mycoses represented with an X encompassed in a circle. Three patients tested were Coccidioides culture positive and are denoted as yellow squares with a black center. A legend with all symbols and their meaning is included in the upper right portion of the graph. The cutoff for positivity is designated on the graph as a dotted line.

Fig 3

(A) Density units for 143 human serum samples tested by CTS1 LFA. The group on the left of the graph includes patients who were positive by CF and/or ID: samples that were positive by both CF and ID are represented by black circles; samples positive by CF but negative by ID (IgG and/or IgM) are shown as grey squares; and samples negative by CF but positive by ID (IgG and/or IgM) are shown as blue diamonds. The group in the middle of the graph includes patients who were negative by CF and ID: samples that were positive by EIA IgG are shown as green triangles and samples negative by EIA are shown as black inverted triangles. The group on the right of the graph shows patients with positive serology to other endemic mycoses represented with an X encompassed in a circle. Three patients tested were Coccidioides culture positive and are denoted as yellow squares with a black center. A cutoff for positivity at 46,000 density units is shown as a blue dashed line. (B) Density units for 50 canine serum samples tested, separated by ID results for endemic samples, and nonendemic samples on the right of the graph. (C) Density units for 33 macaque plasma samples tested, separated by ID results. (D) Density units for 15 dolphin serum samples tested, separated by ID results. Purple squares represent longitudinal samples from the same dolphin collected over 5 years. Dolphins with other mycoses are designated with an X encompassed in a circle. Statistical analysis was performed using Wilcoxon rank sum test with Bonferroni correction (*P ≤ 0.01 and **P < 0.001).

Fig 4

Observed relationship between antibody titer by CF or ID and LFA density units in (A) human, (B) canine, (C) macaque, and (D) dolphin samples. For each titer group, a line represents mean density units. Gray squares in (A) represent samples that were positive by CF but negative by ID.

Fig 5

Visual comparison of sōna Coccidioides Ab LFA and CTS1 Ab LFA. (A) Positive (+) and negative (−) test results using controls included in the sōna Coccidioides Ab LFA kit. (B) Positive (+) and negative (−) test results using VF-positive and VF-negative patient serum, respectively, in the CTS1 Ab LFA. For each test in (A) and (B), control lines are denoted with a “C” and test lines are denoted with a ”T”. (C) Examples of different results produced by the sōna LFA and CTS1 LFA for four patients. Sample H2410 is a nonendemic sample recorded to have positive Histoplasma and negative Coccidioides serology. H2410 produced a positive result by the sōna LFA and a negative result by the CTS1 LFA (14,718 density units). Samples S842 (CF titer of 1:8) and S907 (CF titer of 1:32) both produced a negative result by the sōna LFA and a positive result by the CTS1 LFA (165,119 and 371,565 density units, respectively). Sample S932 (CF titer of 1:128) produced a positive result by both assays, with a darker line shown by the CTS1 LFA (301,489 density units). Sample S1090 (CF titer negative, Coccidioides culture-positive) produced a positive result by the sōna LFA and a negative result by the CTS1 LFA (11,693 density units). Density units for the CTS1 LFA are shown above each test. Diagnostic results by Coccidioides ID and CF are shown below each test.