Specimens

A panel of 371 archived, deidentified frozen serum specimens was prepared at the CDC (Atlanta, Georgia, USA). Of these, 127 syphilis specimens were obtained from the CDC’s syphilis serum repository bank,18 originally procured from the Association of Public Health Laboratory (APHL, Silver Spring, Maryland, USA) through an ongoing collaborative agreement beginning in 2015 . Syphilis stage of those specimens was reported by the participating local and state public health laboratories to the APHL from the test request form. Of the syphilis specimens, suspected disease stage was reported that includes 13 as primary, 43 as secondary, 28 as early latent and 43 as late latent. The remaining 244 specimens included in our testing were syphilis non-reactive (NR) collected from the Georgia Public Health Laboratory (Atlanta, Georgia, USA) in 2016. All specimens were deidentified of any personally identifiable information and tested at the CDC, independent of their previously reported results using RPR (Arlington Scientific, Springville, Utah, USA), TP-PA assay (Fujirebio Diagnostics, Malvern, Pennsylvania, USA) and Trep-Sure EIA (Trinity Biotech USA, Jamestown, New York, USA) as per the respective manufacturer’s package insert. These specimens were also tested using the NT-MAA prototype.

Affinity-purified anticardiolipin antibody (ACA) preparation

The ACAs of IgG and IgM classes were purified from a reactive human syphilitic serum specimen (RPR titre 1:128; Plasma Service Group, Huntingdon Valley, Pennsylvania, USA) using an in-house method of affinity chromatography. Briefly, RPR reactive serum was subjected to ammonium sulfate precipitation and dialysis followed by application of the dialysed fractionate to a cardiolipin-affinity column. The affinity column was prepared by linking cardiolipin to carboxyLink coupling resin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) using a method described by Castro et al.17 The affinity-purified fractionate was then subjected to Protein-A matrix separation (Affi-Gel Protein A Resin, Bio Rad Laboratories, Hercules, California, USA), which after column washing and elution, yielded separate distinct peaks for IgM and IgG ACA. [Deutsch JW, Castro AR, Shukla MR, et al. Development of an enzyme-linked immunosorbent assay (EIA) for the quantification of nontreponemal immunoglobulins IgG and IgM in the sera and plasma of patients with syphilis. 2018]. The ACA were concentrated by ultrafiltration using a 10 KDa membrane (Ultracel 10 kD, Millipore, Bedford, Massachusetts, USA). The purity of ACA was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concentration determined using the radial immunodiffusion test (Kent Laboratories, Bellingham, Washington, USA) following the manufacturer’s directions. The reactivity of purified ACA was confirmed by RPR testing, then prepared as aliquots of 0.25 mL volume per tube and stored at −30 ºC until use.

Cardiolipin modification method

Cardiolipin was oxidised by a method described by Castro et al.17 Briefly, 100 mg of cardiolipin powder (Avanti Polar Lipids, Alabaster, Alabama, USA) was dissolved in 2 mL of t-butanol (Sigma-Aldrich, St. Louis, Missouri, USA) followed by addition of 60 mg of sodium m-periodate (Sigma-Aldrich) and 8 mg of potassium permanganate (Thermo Fisher Scientific) salts in aqueous solution as described. The reaction was continued for 24–48 hours under inert argon gas atmosphere and then stopped with 80 mg of sodium bisulfite (Sigma-Aldrich). The solution was centrifuged at 1000×g for 5 min and the t-butanol phase was then dialysed with 10 mM phosphate buffer, pH 8.0, followed by lyophilisation.

Cardiolipin coupling to magnetic microbeads

A 1 mL volume of aminated magnetic microbeads from the supplier (Chemicell, Berlin, Germany) was concentrated using a magnetic separator (Chemicell) followed by supernatant removal. Concentrated microbeads were suspended to a 1 mL total volume using 10 mM phosphate buffer, pH 5.6. Microbeads were again concentrated and resuspended using the phosphate buffer as described above (two washes total) to obtain a 1 mL volume that was coupled to 10 mg/mL of oxidised/activated cardiolipin. The functional carboxyl group of cardiolipin was activated using 10 mg of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (Sigma-Aldrich) and 10 mg of sulfo-N-hydroxysulfosuccinimide (Thermo Fisher Scientific) in a glass vial.17 The coupling was continued for 2 hours on a rocker at room temperature (21ºC–25ºC). The beads were washed twice with phosphate buffer and then blocked with 10% bovine serum albumin (BSA, Sigma-Aldrich) prepared in deionised water. The cardiolipin-coupled magnetic microbeads were stored at refrigerated temperature (2ºC–8ºC).

Test method for NT-MAA

For a given serum specimen, 25 µL of 1× phosphate-buffered saline (PBS) and 25 µL of serum (1:2) were dispensed in a single well of a clear round bottom 96-well microplate. In a second well, 50 µL of cardiolipin-coupled magnetic microbeads (prediluted to 1:30 in 1× PBS containing 1% BSA) was dispensed, followed by the addition of 10 µL of the 1:2 diluted serum specimen to the same well. The microplate was placed on a plate shaker at 1000 rpm for 1 min to mix, and then incubated on a vibration-free surface for 90–120 min at room temperature (21ºC–25ºC). Optionally, the room temperature incubation can be extended overnight and read thereafter. A test was interpreted as reactive (R) when visible clumps were formed as a result of agglutination lattice formation among ACAs and the cardiolipin-coupled magnetic microbeads. A diagram representing NT-MAA reactivity patterns is shown in figure 1. A nonreactive (NR) sample is defined as a compact button-shaped pattern in the centre of a microwell.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Representation of the reactivity patterns observed in NT-MAA (top) and RPR (bottom) tests using a higher reactive syphilis specimen diluted in normal human serum (1:8 to 1:256). NT-MAA, non-treponemal magnetic particle-based agglutination assay; RPR, rapid plasma reagin; R, reactive; Rm, minimally reactive; NR, non-reactive.

Data analysis

Sensitivity and specificity of tests were calculated using the following standard formula:7

% Sensitivity = [True positive/(True positive+False negative)] × 100

% Specificity = [True negative/(True negative+False positive)] × 100

The sensitivity of tests was calculated using reported syphilis staged specimens and were considered as true positive reference. The true positive number obtained by a respective test is divided by the same, plus false-negative results and were expressed as a percentage to determine sensitivity. For specificity calculation, syphilis NR specimens were included and considered as the true-negative reference, where the true-negative number obtained by a respective test is divided by the same, plus false-positive results followed by percentage expression as shown in the formula above. The CI was determined using Wilson interval estimates. The per cent agreement and kappa value between RPR and NT-MAA were determined using GraphPad web-based software (La Jolla, California, USA). Affinity-purified ACA of IgG and IgM classes were tested separately to determine analytical sensitivity; end-point titre was determined by twofold serial dilution and testing to determine lower limit of detection of both NT-MAA and RPR. The reproducibility of NT-MAA was assessed using serial dilutions of a higher titre RPR reactive serum diluted in normal human serum (from 1:1 to 1:512); prepared dilutions represent specimens with variable reactivity. Intra-assay reproducibility of NT-MAA was performed by testing each dilution 10 different times in a single assay run, whereas interassay reproducibility was executed by testing all dilutions over five separate assay runs. Qualitative results defined as being R, minimally reactive (Rm) or NR were recorded during interpretation. Internal controls defined as syphilis R, Rm and NR were used at the time of testing for quality assurance.