Rapid Amp NP Test: A Game-Changer for Early Detection of  Aminopenicillin Susceptibility/Resistance  in UTI Pathogens

Antibiotic resistance has quietly become one of the most serious threats facing modern medicine and now claims hundreds of thousands of lives each year due to infections that no longer respond to the drugs we’ve relied on for decades. Among these infections, urinary tract infections (UTIs) stand out not only because they are so common—particularly in women—but also because they’re becoming increasingly resistant to standard treatments. Most UTIs are caused by Escherichia coli and other members of the Enterobacterales order which were once easily managed with basic antibiotics like aminopenicillins, but that’s no longer the case. The rise of resistance, largely driven by the widespread emergence of enzymes known as β-lactamases, has forced clinicians to fall back on broader-spectrum antibiotics. While that shift is often necessary, it contributes to a larger problem: it accelerates resistance by applying greater selective pressure on bacteria. This puts healthcare providers in a difficult spot. On the one hand, time is of the essence when treating infections, especially in vulnerable patients. On the other hand, identifying whether a bacterium is resistant—especially due to β-lactamase production—usually takes at least 18 to 24 hours with traditional culture-based methods. That’s too long in most clinical settings, so broad-spectrum antibiotics are frequently prescribed upfront, often based on little more than educated guesses. This approach, while understandable, has unintended consequences. It can lead to overtreatment, misuse of powerful antibiotics, and diminished effectiveness of drugs we may desperately need in the future. There’s a clear need for diagnostic tools that can quickly and accurately detect resistance, ideally from the original patient sample or from early-stage bacterial cultures. But the current diagnostic tools are inefficient because they are complex, expensive, or tailored to specific resistance genes. To address this critical gap, a research team led by Professor Patrice Nordmann at the University of Fribourg, along with collaborators Nicolas Helsens, Nicolas Kieffer, Camille Tinguely, Gilbert Greub, and Laurent Poirel, developed and validated a new and novel diagnostic test called the Rapid Amp NP test. Published in Microbiology Spectrum, their study set out to create a diagnostic solution that is not only fast—delivering results in under two hours—but also affordable, practical, and accessible for everyday clinical use. Unlike gene-based methods, this innovative test detects actual enzymatic activity and can offer a more direct and functional readout of β-lactamase presence. It will contribute to identifiy at least aminopenicillin susceptibility or resistance in worldwide context of reintroduction to this class of antibiotics to treat urinary tract infections.

To evaluate the accuracy and practical potential of their new diagnostic tool, the researchers assembled a panel of 112 bacterial isolates collected from real clinical sources, including urine, blood cultures, and rectal swabs. They wanted to capture a broad representation of organisms commonly linked to urinary tract infections including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Importantly, they included both β-lactamase-positive and -negative strains which allowed for a rigorous evaluation of how well the test could distinguish resistant bacteria from susceptible ones. The authors designed the experimental process itself to reflect a real-world clinical context where they simulated early infection conditions, each strain was grown in broth cultures at bacterial concentrations of 10⁵ and 10⁴ CFU/mL—levels frequently found in urine during a UTI. After a relatively short 90-minute incubation, the bacterial suspensions were filtered and lysed directly on a nylon membrane. This step enabled the application of nitrocefin, a chromogenic cephalosporin that shifts from yellow to red when it encounters β-lactamase activity. The test’s readout was as simple as observing that color change, providing a visually intuitive indication of resistance.

The initial results were quite promising. At the 10⁵ CFU/mL threshold, the Rapid Amp NP test correctly identified β-lactamase activity in 87.6% of the resistant strains tested. Most notably, every single strain producing extended-spectrum β-lactamases (ESBLs) or carbapenemases tested positive—many within just 20 minutes. These particular enzymes are among the most clinically worrisome forms of resistance, so achieving 100% detection in this category strongly supported the test’s clinical utility. The specificity of the test was equally impressive and none of the 15 β-lactamase-negative strains produced false-positive results. Still, the test wasn’t flawless. When they dropped the bacterial concentration to 10⁴ CFU/mL, the detection rate dropped to 56.7%. This decline in sensitivity likely reflects the fact that some strains, particularly those producing narrow-spectrum β-lactamases, generate only small amounts of enzyme or express them at low levels, making detection more difficult when fewer bacteria are present. Despite this, the test still picked up resistance in about 63% of E. coli isolates at this lower concentration.

To further understand the test’s behavior, the authors compared it to a traditional nitrocefin disc assay which is the method commonly used in clinical microbiology labs. Interestingly, some S. aureus strains, including methicillin-resistant ones, were positive using the classic disc method but undetectable with the Rapid Amp NP test. This wasn’t entirely surprising, as it reflects a known issue: nitrocefin is less sensitive for detecting β-lactamase activity in S. aureus, particularly in suspension-based assays. While this revealed a limitation, it also highlighted the importance of using the test alongside species identification, since not all bacteria behave the same way in diagnostic workflows.

The research team also took a closer look at how the type of β-lactamase influenced the speed of detection. They observed that strains producing ESBLs or carbapenemases generally triggered a color change much faster—within 5 to 30 minutes—while strains expressing narrow-spectrum enzymes like TEM-1 could take up to two hours. This difference likely stems from variations in enzyme concentration or catalytic efficiency, and it underscored the need to check the test at multiple time points rather than relying on a single readout. One of the more intriguing findings came from their analysis of cefazolin resistance. Cefazolin, though considered a narrow-spectrum cephalosporin, is still widely used in pediatric UTI cases. Among the cefazolin-resistant, β-lactamase-producing strains tested, an impressive 92% yielded a positive result at the 10⁵ CFU/mL level. This observation suggests that the test might have broader diagnostic applications beyond just detecting aminopenicillin resistance—it could also help identify cephalosporin resistance, particularly in patient populations where treatment options are more limited.

Finally, Professor Patrice Nordmann and his colleagues addressed a practical challenge: whether the test could be used directly on patient urine samples without pre-culture. While this proof-of-concept study didn’t yet reach that goal, the researchers conducted early experiments which indicated that dead bacteria might continue to release β-lactamases over time in the bladder and potentially boosting the signal. This is important because it opens the door to future refinements aimed at improving sensitivity and making the test even more point-of-care friendly.

In conclusion, Professor Patrice Nordmann and his colleagues successfully developed Rapid Amp NP test as a simple, rapid, and low-cost diagnostic tool to detect β-lactamase activity, particularly in bacteria responsible for urinary tract infections. This is especially important given how slowly traditional diagnostics work. In many clinical settings, the standard approach to identifying antibiotic resistance takes nearly a full day or more, leaving clinicians to rely on empiric therapy in the meantime. That usually means prescribing broad-spectrum antibiotics, which, while well-intentioned, contributes to the growing problem of antibiotic resistance by applying unnecessary pressure on bacterial populations. We believe what stands out about the diagnostic innovation of Rapid Amp NP test is its emphasis on real-world usability. It doesn’t require advanced instrumentation or costly reagents, and its readout—a straightforward color change—is designed to be intuitive, even for non-specialists. That means it could potentially be used not just in hospitals or large diagnostic labs, but in outpatient clinics and general practice settings. In places where access to complex molecular tools is limited or entirely unavailable, this kind of simplicity could be transformative. Moreover, if future larger clinical trials confirms that the test can be used directly on urine samples, it could become a routine part of point-of-care testing and allow healthcare providers to make informed treatment decisions during a single patient visit. The clinical implications are significant. With the ability to quickly determine whether a UTI-causing organism produces β-lactamase, clinicians could confidently prescribe narrow-spectrum antibiotics like amoxicillin when appropriate. This would help preserve the efficacy of more powerful drugs for patients who truly need them, while reducing side effects and unnecessary antibiotic exposure for everyone else.