Cross-Reactivity of GeneXpert MTB/RIF Probe C with Nocardia Species as a Suggestive Ancillary Indicator.

Abstract

Background: The slow-growing, fastidious nature of Nocardia species makes rapid molecular tools essential for diagnosis. Although the GeneXpert MTB/RIF assay targets Mycobacterium tuberculosis (MTB), the cross-reactivity of its Probe C with Nocardia has not previously been characterized.

Methods: We retrospectively analyzed 14,141 non-duplicate clinical samples from February 2020 to June 2023. Probe C reactivity was first assessed against Nocardia culture results. To investigate the molecular basis, we subsequently compared the rpoB target region of Probe C with the genomes of 1082 Nocardia strains from the NCBI database.

Results: Probe C showed species-specific reactivity, failing to detect many clinically prevalent species. Among culture-positive Nocardia isolates, Probe C detected 10 of 17 N. cyriacigeorgica (Ct range: 32.8-39.2), 1 of 2 N. asiatica (Ct: 39), and 2 of 8 N. abscessus (Ct range: 30.5-38.7). Notably, the most frequently isolated species, N. farcinica (59 isolates), showed no Probe C reactivity. The overall sensitivity of Probe C among all culture-confirmed nocardiosis cases was only 13.4% (13/97); even among the subset of cross-reacting species, it reached only 48.1% (13/27), highlighting its inadequacy as a standalone diagnostic test. In the full MTB-negative cohort, the Probe C-positive rate was just 0.27% (38/14,141), with 25 of those 38 signals lacking culture confirmation. Genome alignment confirmed 100% identity between the rpoB sequences of reactive Nocardia strains and the Probe C target region.

Conclusion: Probe C is the first MTB-targeted tool demonstrated to cross-react with Nocardia via rpoB homology, with genomic evidence ruling out other genetic sources. However, given the low overall sensitivity of Probe C among all culture-confirmed nocardiosis cases, the restricted species spectrum, and the lack of molecular confirmation for most positive signals at high Ct values, an isolated Probe C-positive result in an MTB-negative sample should be regarded as a suggestive, non-diagnostic ancillary hint that warrants Nocardia-specific confirmatory testing rather than as a diagnostic finding.