## 1 Introduction  
Since the 1960s, microbiology has undergone significant evolution, with each decade bringing notable advancements. From the late 1990s to the early 2000s, molecular diagnostic technologies such as polymerase chain reaction (PCR) were widely implemented in the medical field. The introduction of Nucleic Acid Amplification Tests (NAATs) in microbial diagnostics addressed the limitations of traditional, time-consuming culture methods, enabling more rapid and specific detection of pathogens. This development led to the emergence of culture-independent diagnostic tests (CIDTs), specifically designed to overcome the challenges associated with traditional culture techniques [1]. The early 2000s marked a pivotal shift with the advent of molecular methods, which facilitated the development of CIDTs capable of detecting multiple pathogens from a single specimen, thereby bypassing the need for culture. By the late 2000s and into the 2010s, the incidence of infections involving multiple pathogens increased, creating a demand for diagnostic technologies capable of simultaneously detecting a broad spectrum of pathogens for public health and biodefense purposes. This need drove the development of Highly Multiplexed Microbiological/Medical Countermeasure Diagnostic Devices (HMMDs) [2]. Building on CIDT technology, HMMDs introduced advanced multiplex approaches, allowing for the simultaneous diagnosis of multiple pathogens (Figure 1). Figure 1: Advances in molecular diagnostics for infectious disease detection. The figure shows the progression from traditional culture methods to advanced molecular diagnostics, allowing for direct, multiplex detection of pathogens without the need for culture. With the introduction of PCR-based Nucleic Acid Amplification Tests (NAAT) in the 1990s, diagnostics evolved to include Culture-Independent Diagnostic Tests (CIDT) and array-based methods. This advancement continued into the 2020s with the development of Highly Multiplexed Microbiological/Medical Countermeasure Diagnostic Devices (HMMDs), enabling comprehensive multiplex technologies to simultaneously diagnose multiple pathogens. In January 2013, four HMMDs for detecting acute gastroenteritis (AGE) pathogens received approval from the Food and Drug Administration (FDA), resulting in their rapid integration into microbiology laboratories. These devices represented a significant advancement in the field, meeting the growing need for comprehensive and efficient pathogen detection in clinical settings [1; 3; 4; 5]. Today, several FDA-approved multiplex nucleic acid-based assays are available for detecting foodborne pathogens associated with AGE. These assays include the BIOFIRE FILMARRAY Gastrointestinal Panel (bioMerieux, Marcy-I'Etoile, France), xTAG Gastrointestinal Pathogen Panel (Diasorin, Saluggia, Italy), BD MAX Enteric Bacterial Panel (Becton Dickinson, Franklin Lakes, NJ, USA), QIAstat-Dx Gastrointestinal Panel 2 (QIAGEN, Hilden, Germany), and Biocode Gastrointestinal Pathogen Panel (Applied BioCode, Santa Fe Springs, CA, USA). These tests are capable of simultaneously detecting between 9 and 15 pathogens, including bacteria, viruses, and parasites, within approximately 5 h (Table 1, refer to Supplemental Table S1 for more detailed information). According to the Centers for Disease Control and Prevention (CDC), the use of HMMDs for AGE pathogen detection has surged over the past decade. Healthcare providers are increasingly adopting these tests and DNA-based syndromic panels due to their rapid results and user-friendly nature compared to conventional culture methods [6]. By 2023, HMMDs were used in 78% of bacterial infection diagnoses, with 46% relying solely on these tests [7]. HMMDs offer several advantages, including faster turnaround times for targeted treatment, the ability to detect or rule out multiple pathogens from a single test, and potentially higher sensitivity compared to traditional culture methods. They also hold promise in resource-limited settings.