## 4 Discussion  
With advancements in molecular diagnostics, including technologies like HMMDs and the anticipated development of NGS, microbiology has entered a new era of data-driven insights. HMMDs, such as the Luminex xTAG GPP, enable rapid, multiplex pathogen detection directly from patient samples, providing valuable information on potential infections. These technologies have demonstrated significant utility in reducing turnaround time and detecting co-infections, as evidenced by prior studies highlighting their effectiveness across various bacterial, viral, and parasitic pathogens, particularly those difficult to culture. However, as molecular techniques generate vast amounts of nucleic acid data, they bring forth interpretive challenges that require more nuanced frameworks to ensure clinically meaningful outcomes.  
Our study sought to evaluate the impact of manufacturer-introduced changes to the _Salmonella_ MFI cutoff in the Luminex xTAG GPP, illustrating these interpretive challenges. After adjusting the cutoff, the rate of false positives in HMMD-positive but culture-negative cases slightly decreased from 40.6% to 38.4%. However, this adjustment also resulted in one culture-confirmed positive case (3.7%) being incorrectly reported as negative. The patient in this false-negative case, a 6-year-old girl with acute diarrhea and hematochezia, had a probe 1 MFI value below the newly established cutoff of 300, leading to a negative interpretation. This example highlights a critical issue: while false-positive results may arise from residual nucleic acids or cross-reactivity inherent in molecular testing, false-negative results may stem from low bacterial load or limitations in assay sensitivity [11; 12]. The cutoff adjustment appeared to slightly refine the positive/negative boundary, but it did not improve diagnostic accuracy. Instead, this modification reflects a horizontal shift in threshold criteria, emphasizing the limitations of binary interpretation in molecular diagnostics.  
It is noteworthy that the Luminex xTAG GPP does not provide detailed guidance on the principles behind positive judgment or the rationale for probe settings, leaving laboratories dependent on manufacturer-defined thresholds. This strict adherence to binary interpretation restricts diagnostic flexibility and, as demonstrated in this study, can lead to both false-positive and false-negative results. Such cases emphasize the need for more refined interpretive strategies, including the potential introduction of an \"indeterminate\" category for borderline results, to bridge the diagnostic gaps in molecular testing.  
While the broader utility of the Luminex xTAG GPP remains indisputable [13]--particularly in its ability to rapidly detect a wide range of pathogens--our study highlights  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Stool** & **Stool** & **Stool Culture Not** & \\  & **Culture-Positive** & **Culture-Negative** & **Tested** & _p-Value_ \\ \hline Probe 1 & 2264.96 \(\pm\) 1273.77 & 1232.49 \(\pm\) 860.18 & 1247.09 \(\pm\) 1209.37 & \textless{}0.005 \\ Probe 2 & 2084.57 \(\pm\) 1310.64 & 1521.54 \(\pm\) 1040.57 & 1379.59 \(\pm\) 1343.62 & 0.004 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Comparison of probe-specific MFI differences (mean \(\pm\) standard deviation) based on stool culture results for _Salmonella_-positive samples, regardless of MFI threshold adjustments in the xTAG Gastrointestinal Pathogen Panel.  
the importance of reevaluating the interpretive frameworks applied to HMMDs. This need extends beyond the specific case of the Luminex xTAG GPP to the broader challenges posed by binary molecular interpretations in clinical microbiology. As the integration of NGS into diagnostic workflows becomes increasingly imminent, it is critical to develop thoughtful and adaptive approaches that enhance the clinical utility of molecular data.