The unsung hero: ntnh gene as complementary botulism marker

Botulism is a rare but severe neurological disease caused by botulinum neurotoxins (BoNTs). Standard diagnostic methods including the mouse bioassay (SMB) and bont gene type-specific PCR, are often limited by the high genetic diversity among bont subtypes, which can lead to false-negative results. The nontoxin-nonhemagglutinin (ntnh) gene is highly conserved and exclusively co-located with the bont gene complex. Thus, this study evaluates the use of ntnh gene as a complementary diagnostic tool for botulism and assesses its association with BoNT types. The ntnh gene was detected in a prospective BoNT-diagnostic group (n=88) and a BoNT-historical group (n=54). Toxin cluster proteins were identified in GenBank and RefSeq Clostridium proteomes using MMSeqs2. ntnh gene detection reinforced positive results from SMB or bont gene tests in 26 cases (35.62% of the total confirmed cases) of foodborne and infant botulism. In two foodborne cases from the BoNT-diagnostic group, the ntnh gene was detected despite negative results from both SMB and bont gene tests, highlighting its potential to identify missed cases. An in silico analysis of 3,250 RefSeq and 2,494 GenBank annotated Clostridium proteomes was conducted. respectively. So, NTNH showed a high co-presence pattern with BoNT. Moreover, NTNH sequences were far more conserved than BoNT sequences in inter-type comparisons (67.2 vs.39.7), which highlights its applicability as a disease biomarker. The ntnh gene analysis is a valuable complementary tool enhancing the diagnosis of botulism. The study highlights the need for clear guidelines to interpret positive ntnh results when direct toxin or bont gene confirmation are negative.