Epsilometer test (E test)- Principle, Procedure, Results, Advantages

Antimicrobial resistance is a major public health concern that threatens the effectiveness of existing treatments for various infections. One of the key steps in managing antimicrobial resistance is to determine the susceptibility of microorganisms to different antibiotics. This can help clinicians to choose the most appropriate therapy and prevent the spread of resistant strains.

There are various methods for testing antimicrobial susceptibility, such as disk diffusion, broth dilution, agar dilution, and automated systems. However, these methods have some limitations, such as being time-consuming, labor-intensive, requiring specialized equipment or media, or having low accuracy or reproducibility.

The Epsilometer test (E-test) is a novel method that overcomes some of these limitations and provides a direct quantification of antimicrobial susceptibility of microorganisms. It is defined as the “exponential gradient” method that combines both the dilution and diffusion principles of antibiotic testing .

The E-test uses rectangular plastic strips that have a predefined, continuous and exponential gradient of antibiotic concentration along one side. The other side of the strip has a numeric scale that indicates the drug concentration in micrograms per milliliter (µg/ml). The strip is applied to an inoculated agar plate and incubated for a specified time. The antibiotic diffuses from the strip into the agar and creates an inhibition zone around it. The minimum inhibitory concentration (MIC) is the lowest concentration of the antibiotic that inhibits the visible growth of the microorganism. The MIC can be read from the scale where the edge of the inhibition zone intersects the strip .

The E-test is a cost-effective, simple and reliable method that can be used for a wide range of antibiotics and microorganisms. It can also detect low levels of resistance or specific phenotypes of resistance that may be missed by other methods. The E-test has been widely used in clinical microbiology laboratories, research settings and epidemiological studies .

The E-test has several objectives that make it a useful and convenient method for determining the antimicrobial susceptibility of microorganisms. Some of the main objectives are:

• To determine the MIC of fastidious, slow-growing or nutritionally deficient micro-organisms that are difficult to test by other methods .
• To detect and confirm low levels of resistance of a specific antimicrobial phenotype resistance, such as vancomycin-resistant enterococci or methicillin-resistant Staphylococcus aureus .
• To determine and demonstrate the MIC value of the given antibiotic to given micro-organism, which can help in choosing the appropriate dosage and duration of treatment .

The E-test can also be used to investigate the synergistic potential of combination therapies, such as amphotericin B and flucytosine for Cryptococcus neoformans infections. However, this requires careful interpretation of the results and knowledge of the interactions between different antibiotics.

The E-test is based on the principle of combining both the dilution and diffusion of antibiotics into the agar medium. The E-test uses a rectangular plastic strip that has a predefined, continuous and exponential gradient of antibiotic concentration along its length. One side of the strip contains the antimicrobial agent concentration gradient and the other side contains a numeric scale that indicates the drug concentration in micrograms per milliliter (µg/ml).

When the E-test strip is placed on an inoculated agar plate, there is an immediate release of the antibiotic from the plastic carrier surface into the agar surface. The antibiotic diffuses radially from the strip into the agar, creating a concentration gradient that decreases exponentially from the strip towards the edge of the plate. After incubation, bacterial growth becomes visible on the plate, and a symmetrical inhibition ellipse along the strip is seen.

The minimum inhibitory concentration (MIC) of the antibiotic for the test organism is read from the scale at the point where the lower edge of the ellipse intersects with the strip. The MIC is defined as the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism. The MIC values of the bacteria can be interpreted as susceptible, intermediate or resistant according to the clinical and laboratory standards institute (CLSI) guidelines.

The E-test is a simple and reliable method that provides a direct quantification of antimicrobial susceptibility of microorganisms. It can be used for a wide range of antibiotics and microorganisms, including fastidious, slow-growing or nutritionally deficient ones. It can also detect and confirm low levels of resistance or specific phenotypes of resistance.

The E-test is a simple and convenient method that requires minimal training and equipment. The following steps describe the general procedure for conducting the E-test:

• Inoculum preparation: The bacterial strain to be tested should be grown on a suitable culture medium and isolated as single colonies. Three or four colonies of the same strain should be picked with a sterile loop and emulsified in a tube of sterile saline. The turbidity of the inoculum should be adjusted to match the 0.5 McFarland standard, which corresponds to approximately 1.5 x 10^8 CFU/ml.
• Inoculation in Muller Hinton Agar: A sterile cotton swab should be dipped into the inoculum and rotated against the inside of the tube above the fluid level to remove excess liquid. The swab should then be used to streak the entire surface of a Mueller Hinton Agar (MHA) plate by rotating the plate at an angle of about 60 degrees. The inoculation should be completed by running the swab around the edge of the agar. The lid of the plate should be left slightly open for 5 minutes (but not more than 15 minutes) to allow any excess moisture to be absorbed before applying the E-test strips.
• Application of E-test strips: The E-test strips should be removed from the freezer (-20°C) at least 30 minutes before use and opened by cutting along the broken line of the package. Using sterile forceps, one E-test strip should be applied to each inoculated agar plate, with the `E` end at the edge of the plate and the scale facing upwards. The strip should be gently pressed onto the agar surface to ensure good contact. Care should be taken not to touch the edge of the plate or move the strip once applied. If multiple strips are used on a single plate, they should be spaced at least 24 mm apart from each other and from the edge of the plate.
• Incubation: The plates should be incubated at 37°C for 18-24 hours in an appropriate atmosphere, depending on the bacterial species tested.

• After the incubation, the tests are read by viewing the strips from the top of the plate. A symmetrical inhibition ellipse is produced along the strip on the plate .
• Read MIC at the point where the ellipse intersects the scale on the E-test strip. If a MIC value between two twofold dilutions is seen, always round up to the highest value .
• Read the MIC value at complete inhibition of all growth. If the intersect differs on either side of the strip, read the MIC as the greater value .
• MIC values of the bacteria should be interpreted as S (Susceptible), I (Intermediate) or R (Resistant) by comparing the breakpoint values of each antibiotic with the criteria recommended and given by CLSI guidelines .
• An example of E-test result interpretation is shown below:

• When conducting the E-test, it is important to follow aseptic procedures and precautions against microbiological hazards when handling bacterial specimens.
• The E-test should be used strictly according to the procedures described by the manufacturer and the FDA. Any deviation from the recommended protocol may affect the accuracy and reliability of the test results.
• One should be very careful while placing the E strip on the plate containing the bacterial suspension to not touch the edge of the plate or any other surface. This may contaminate the strip or the agar and interfere with the diffusion of the antibiotic gradient.
• The E-test results should be read only if a good inhibition ellipse is visible on the plate. If there is no clear zone of inhibition or if there is any irregularity in the shape of the ellipse, the test should be repeated or confirmed by another method.
• When using multiple strips on a single plate, the strips should be placed at least 15 mm apart from each other and from the edge of the plate. This will prevent any overlap or interference between the antibiotic gradients of different strips.

The E test has several advantages over other methods of antimicrobial susceptibility testing, such as disk diffusion and broth dilution. Some of these advantages are:

• It is very easy to perform, requires minimal training for test performance, and easy to execute. The E test strips are ready to use and do not need any preparation or calibration. The procedure is simple and involves only inoculating the agar plate, applying the strip, and incubating the plate. The results are easy to read and interpret by using the scale on the strip.
• Contamination can be easily recognized. Unlike broth dilution methods, where contamination may not be visible until the end of the incubation period, the E test allows for immediate detection of any contamination on the agar plate. Any growth outside the inhibition ellipse or along the strip indicates contamination and invalidates the result.
• It is a less time-consuming and very convenient method of the determination of the Minimum Inhibitory Concentration (MIC) and applicable to a wide array of drugs and microorganisms. The E test can provide MIC results within 18-24 hours for most bacteria and fungi, whereas other methods may take longer or require additional steps. The E test can also be used for testing a variety of antimicrobial agents, including antibiotics, antifungals, antivirals, and antiparasitics. Moreover, the E test can be applied to different types of microorganisms, such as aerobic and anaerobic bacteria, fastidious and non-fastidious organisms, yeasts and molds, and mycobacteria.
• It is useful to detect some phenotypes resistance. The E test can help identify some specific mechanisms of resistance that may not be detected by other methods. For example, the E test can detect heteroresistance in methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin-resistant enterococci (VRE). The E test can also detect inducible resistance in some gram-negative bacteria that produce beta-lactamases.
• It is an adequate method to detect potentially resistant strains to Amphotericin B. The E test has been shown to be reliable and accurate for testing the susceptibility of Candida species and Aspergillus species to Amphotericin B, which is a widely used antifungal agent. The E test can also differentiate between susceptible and resistant strains of these fungi based on their MIC values.
• It helps to confirm or detect low-level or new resistance mechanisms. The E test can provide precise MIC values that can reveal subtle differences in susceptibility among strains of the same species or among different species. This can help confirm or detect low-level resistance that may not be apparent by other methods. The E test can also help identify new resistance mechanisms that may emerge due to genetic mutations or horizontal gene transfer.
• It can be used to investigate any synergistic potential of combination therapies. The E test can be used to evaluate the effect of combining two or more antimicrobial agents against a given microorganism. By placing two or more strips on the same agar plate at different angles or distances, the interaction between the drugs can be observed by measuring the shape and size of the inhibition ellipse. The interaction can be classified as synergistic, additive, indifferent, or antagonistic based on the MIC values obtained from the combination compared to those obtained from each drug alone.

• The E-test is not suitable for some microorganisms, such as Cryptococcus neoformans, that do not produce a clear inhibition ellipse .
• The E-test may not be reliable for testing some antibiotics, such as polymyxins and glycopeptides, that have poor diffusion properties or complex interactions with the agar medium.
• The E-test may not be able to detect some resistance mechanisms, such as inducible beta-lactamases or efflux pumps, that require special test conditions or substrates.
• The E-test may not be accurate for testing combinations of antibiotics, as it is difficult to know if they have an additive, synergistic, or antagonistic effect on the microorganism .
• The E-test may be affected by environmental factors, such as temperature, humidity, inoculum size, and incubation time, that can influence the diffusion and stability of the antibiotics.
• The E-test may be costly and require special storage conditions, as the strips are sensitive to light and moisture and have a limited shelf life.

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