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 <i>bont</i> gene type-specific PCR, are often limited by the high genetic diversity among <i>bont</i> subtypes, which can lead to false-negative results. The nontoxin-nonhemagglutinin (<i>ntnh)</i> gene is highly conserved and exclusively co-located with the <i>bont</i> gene complex. Thus, this study evaluates the use of <i>ntnh</i> gene as a complementary diagnostic tool for botulism and assesses its association with BoNT types. The <i>ntnh</i> 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 <i>Clostridium</i> proteomes using MMSeqs2. <i>ntnh</i> gene detection reinforced positive results from SMB or <i>bont</i> 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 <i>ntnh</i> gene was detected despite negative results from both SMB and <i>bont</i> gene tests, highlighting its potential to identify missed cases. An <i>in silico</i> analysis of 3,250 RefSeq and 2,494 GenBank annotated <i>Clostridium</i> 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 <i>ntnh</i> gene analysis is a valuable complementary tool enhancing the diagnosis of botulism. The study highlights the need for clear guidelines to interpret positive <i>ntnh</i> results when direct toxin or <i>bont</i> gene confirmation are negative.