Abstract
False-positive Neisseria gonorrhoeae (NG) screening results require supplemental nucleic acid amplification testing for confirmation; however, Alinity m multi-Collect media (Alinity media) forms a precipitate when combined with guanidinium-based lysis buffers, interfering with nucleic acid extraction and causing Xpert CT/NG assay failures. This study evaluated a simple pre-extraction method to enable supplemental NG testing from Alinity media. A guanidine hydrochloride pre-extraction step was developed to remove precipitate prior to testing. Sixteen NG-positive urine specimens and a swab dilution series were inoculated into both Alinity media and cobas PCR media as a control. Samples were tested using Xpert CT/NG (NG2 and NG4 targets) and extracted on the MagNA Pure 96 platform for supplemental testing with ResistancePlus GC (RP-GC), targeting opa and porA. Qualitative results from Alinity media and cobas PCR media were in complete agreement, with paired quantification cycle (Cq) values showing high concordance across all targets and internal controls. RP-GC opa and porA targets demonstrated mean Cq differences of -0.45 and - 0.47, respectively, indicating a small but statistically significant earlier Cq for Alinity media (p = 0.004 and p = 0.018). Despite reaching statistical significance, these differences were less than 0.5 cycles and were not considered analytically meaningful. Xpert NG2 and NG4 targets showed negligible, non-significant Cq shifts. These findings demonstrate that the guanidine hydrochloride pre-extraction method effectively removes precipitate from Alinity media and enables reliable supplemental NG testing, with performance equivalent to cobas PCR media across both extraction-based and sample-to-answer platforms.