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Antimicrobial Susceptibility Testing
- Highly standardized methods are essential for all types of antimicrobial susceptibility testing.
- The test results are highly sensitive to variations in inoculum density, media formulation, agar thickness an moisure, potency of the disk, correct storage of the disks, incubation time and how you read and interpretate the inhibition zones. EUCAST- European committee on Antimicrobial Susceptibility Testing CLSI- Clinical and Laboratory Standards Institute "In vivo" means research done on a living organism, while "in vitro" means research done in a laboratory dish or test tube. antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of an organism to an antimicrobial agent at levels corresponding to blood or tissue levels attainable with usually prescribed doses of that agent. 1) susceptible –^ a category that implies that isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the dosage recommended to treat the site of infection is used. 2) intermediate –^ a^ category^ that^ includes^ isolates^ with^ antimicrobial^ agent^ minimal inhibitory concentrations that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates. The intermediate category implies clinical efficacy in body sites where the drugs are physiologically concentrated (eg, quinolones and β-lactams in urine) or when a higher than normal dosage of a drug can be used (eg, β-lactams). This category also includes a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins. 3)
resistant – a category that implies that isolates are not inhibited by the usually achievable concentrations of the agent with normal dosage schedules and/or that demonstrate minimal inhibitory concentrations that fall in the range in which specific microbial resistance mechanisms (eg, β-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies. broth microdilution technique – the method of antimicrobial susceptibility testing that is based on preparation of a liquid broth medium containing various concentrations of antimicrobial agents into which a defined inoculum of microorganisms is inoculated and then incubated and observed for growth. minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test. quality control (QC) – the operational techniques that are used to ensure accuracy and reproducibility in microbiology testing. reference strain – a particular strain of a bacterial species that can be obtained from a microbiological repository, such as the American Type Culture Collection, which exhibits stable genetic properties and yields reproducible minimal inhibitory concentrations when tested in vitro against designated antimicrobial agents. Disk Diffusion Method
- The disk diffusion assay is a qualitative method that relies on the diffusion of antimicrobial agents within agar.
- The extent of this diffusion is measured by determining the diameter of the corresponding inhibition zone, which is used to ascertain the antimicrobial efficacy of the tested material.
- If antibacterial substances have positive antibacterial activity, the growth of bacteria will be inhibited and clear visible inhibitory zones will appear. The zone of inhibition, which is the location free of bacterial colonization, is situated between the perforated area and the bacterial-cultivated region.
- MIC value is determined at the intersection of the strip and the growth inhibition ellipse.
- It is simple to implement; thus, it is routinely used to meet the demands of clinicians.
- This approach becomes costly if numerous drugs are tested. Agar well diffusion method
- Agar well diffusion method is widely used to evaluate the antimicrobial activity of plants or microbial extracts.
- Similarly to the procedure used in disk-diffusion method, the agar plate surface is inoculated by spreading a volume of the microbial inoculum over the entire agar surface.
- Then, a hole with a diameter of 6 to 8 mm is punched aseptically with a sterile cork borer or a tip, and a volume (20– 100 μL) of the antimicrobial agent or extract solution at desired concentration is introduced into the well.
- Then, agar plates are incubated under suitable conditions depending upon the test microorganism.
- The antimicrobial agent diffuses in the agar medium and inhibits the growth of the microbial strain tested. Agar plug diffusion method
- Agar plug diffusion method is often used to highlight the antagonism between microorganisms.
- This method is commonly used to study the antagonism between microorganisms. The first bacterial strain is inoculated onto agar plates in tight streaks. The bacteria will secrete molecules that diffuse in the agar medium; this medium is cut and placed on another agar plate inoculated with another microorganism.
- The procedure is similar to that used in the disk-diffusion method.
- It involves making an agar culture of the strain of interest on its appropriate culture medium by tight streaks on the plate surface.
- During their growth, microbial cells secrete molecules which diffuse in the agar medium. After incubation, an agar-plot or cylinder is cut aseptically with a sterile cork borer and deposited on the agar surface of another plate previously inoculated by the test microorganism.
- The substances diffuse from the plug to the agar medium. Then, the antimicrobial activity of the microbial secreted molecules is detected by the appearance of the inhibition zone around the agar plug. agar dilution technique – the method of antimicrobial susceptibility testing that is based on preparation of agar plates containing various concentrations of antimicrobial agents on which a defined inoculum of microorganisms is inoculated and then incubated and observed for growth
- Nanotechnology-based delivery systems are increasingly being evaluated as viable options for improving therapeutic efficacy by limiting drug degradation, increasing accumulation at infection sites, and reducing toxicity.
- The NPs provide the most effective method of addressing MDR bacteria since they not only act as transporters for natural antibiotics and antimicrobials, but also actively combat bacteria.
- Moreover, loaded NPs have the capacity to safely and effectively transport a wide variety of treatments to the site of infection when they are bonded to their surface or enclosed within a structure.
Antiparasitic drugs Are a crucial component of medical interventions aimed at combating parasitic infections. These medications are specifically designed to target and eliminate parasites that can cause a wide range of diseases in humans and animals. Mechanisms of action Antiparasitic drugs work by interfering with the vital processes or structures of the parasites, ultimately leading to their elimination from the host organism. The specific mechanisms of action vary depending on the type of parasite and the drug class. Some common mechanisms include Enzyme inhibition: Certain antiparasitic drugs disrupt essential enzymes required for the survival and reproduction of parasites. For example, antimalarial drugs like chloroquine inhibit the parasite's ability to break down hemoglobin, leading to its death. Cellular dysfunction: Antiparasitic drugs may disrupt critical cellular functions, such as DNA replication or protein synthesis, causing metabolic dysfunction and parasite death. This mechanism is observed in drugs like metronidazole, used to treat protozoan infections. Nervous system targeting: Some antiparasitic drugs target the nervous system of parasites, affecting their ability to control movement, feed, or reproduce. Ivermectin, for example, acts on the nervous system of parasitic worms, leading to paralysis and expulsion from the host. Types of antiparasitic drugs Antiparasitic drugs encompass a broad range of medications, each targeting specific types of parasites. Some common types include Anthelmintics: These drugs target helminths, which are parasitic worms, including roundworms, tapeworms, and flukes. Anthelmintics may work by paralyzing the worms, inhibiting their energy production, or blocking their nutrient uptake. Examples include albendazole and mebendazole.
While antiparasitic drugs have proven to be effective in combating parasitic infections, they are not without challenges. Some of the key challenges include Drug resistance: Parasites have the ability to develop resistance to antiparasitic drugs over time. This poses a significant challenge in the treatment and control of parasitic infections. Ongoing research and surveillance are necessary to monitor and combat drug resistance. Limited access: In resource-constrained areas, access to antiparasitic drugs may be limited. This can hinder the effective management and control of parasitic infections, particularly in regions where these infections are endemic. Efforts are required to improve access to these medications in such areas. Research and development: The development of new antiparasitic drugs is essential to address emerging challenges, such as drug resistance and the need for more effective treatments. Continued investment in research and development is necessary to expand the arsenal of antiparasitic medications. The significance of antiparasitic drugs in protecting health and well-being cannot be overstated. These medications, with their diverse mechanisms of action, target a wide range of parasites, including helminths, protozoa, and ectoparasites. By disrupting essential processes or structures within the parasites, antiparasitic drugs can eliminate the infection and improve the health and well-being of individuals. They also contribute to the overall public health by preventing the transmission of parasitic diseases and reducing the burden on healthcare systems. Conclusion Antiparasitic drugs are indispensable tools in the fight against parasitic infections. They play a vital role in both human and veterinary medicine by treating and preventing various parasitic diseases. However, challenges such as drug resistance and limited access persist. Continued efforts in education, surveillance, and the development of new antiparasitic drugs are essential to combat parasitic infections and safeguard global health. By understanding the mechanisms of action and the significance of these medications, we can make informed decisions to protect ourselves, our animals, and our communities from the harmful effects of parasitic infections.
Rather than killing or inactivating viruses, antiviral drugs focus on inhibiting viral replication by interfering with specific stages of the viral life cycle. Antiviral drugs use two approaches: targeting the viruses themselves or the host cell factors. Direct virus targets include:
- The inhibitors of virus attachment/entry
- Uncoating inhibitors
- Inhibition of viral replication
- Inhibition of viral protein synthesis
- Inhibition of viral assembly
- Inhibition of release Inhibitors of virus attachment and entry inhibitors
- Drugs of this category target host receptors, co-receptors, or viral spike proteins.
- Drugs that inhibit attachment and virus entry prevent all subsequent steps of the viral replication cycle and virus infection.
- It permits the clearing of virion by the host immune system at the beginning. Example; a drug maraviroc binds CCR5 of a host cell receptor and blocks the viral attachment.
- Drugs like enfuvirtide bind gp41 of the viral envelope and inhibit viral fusion with the host cell membrane.
- Membrane fusion inhibitor drugs target mainly the enveloped viruses. Inhibition of viral uncoating in the host cell
- Some antiviral drugs, like amantadine, prevent uncoating and the release of the viral genome in the cell.
- Amantadine is a narrow spectrum drug that works against only Influenza A.
- Amantadine blocks M2 ion channel function and thereby prevents acidification, dissociation, and uncoating, which prevents the release of nucleic acid from the endosome to the host cell cytosol.
Inhibition of viral replication Drugs inhibiting replication of viral nucleic acid target various sites:
- Polymerase and Reverse Transcriptase Inhibitors Some antiviral drugs target DNA or RNA polymerase to inhibit DNA/RNA replication, e.g., viral DNA polymerase inhibitors, like acyclovir and tenofovir.
- On the other hand, some drugs target the reverse transcriptase (RT) enzyme inhibiting the synthesis of DNA from RNA. Drugs targeting the RT enzyme are efficient and safe, as RT is present only in viruses, not humans. The drugs that inhibit replication is nucleotide/nucleoside analogs and non-nucleotide/nucleoside analogs.
- Nucleotide or nucleoside analogs compete with regular nucleotide/ nucleoside and insert themselves into a growing nucleic acid chain. It stops the process prematurely.
- Nucleoside analogs, like, acyclovir and AZT, lack a 3’OH. Thus, if they get incorporated into a growing nucleic acid strand, all nucleic acid synthesis requires a 3’OH site for adding the next nucleotide.
- Nonnucleoside inhibitors bind non-competitively to the polymerase or reverse transcriptase, impairing its function, e.g., nevirapine.
- Nucleoside RT inhibitors (NRTI) and nonnucleoside RT inhibitors (NNRTI) combine to treat HIV with maximum effect. Integrase Inhibitors
- Integrase inhibitors, like raltegravir, are frequently used. Such drugs prevent the binding of the viral genome to the host genome, which is essential for some viruses. Interferons
- Interferons are low molecular weight proteins produced by virus-infected cells.
Enfuvirtide (HIV) Maraviroc (HIV) binding to gp41. Binds CCR5 of host receptor blocks fusion and entry of the virus. Uncoating inhibitors prevent the release of the genome Amantadine and Rimantadine (Influenza) Inhibit M2 ion channel preventing pH-dependent dissociation of viral proteins, which contain the release of nucleic acid to host cell. Inhibition in viral replication Remdesivir (COVID-19) Favipiravir (COVID-19), Foscarnet (HSV) Acyclovir & Ganciclovir (Herpes) Ribavirin (RSV) Dolutegravir, Elvitegravir and Raltegravur (HIV) Zidovudine(HIV) and Lamivudine (HIV &HBV) Nevirapine & Efavirenz (HIV) RNA dependent RNA polymerase inhibitor, an analog of adenosine nucleotide RNA dependent RNA polymerase inhibitor, an analog of guanosine nucleotide DNA polymerase inhibitor DNA polymerase and Reverse Transcriptase inhibitors (nucleoside analogs) Inhibitor of integrase NRTI (nucleotide analog) NNRTI Viral protein synthesis inhibitors Fomivirsen(CMV) Interferon alfa (HBV, HCV) Antisense therapy for termination. Translation inhibitors prevent viral protein synthesis, which promotes the breakdown of viral components. Inhibitors of Viral Assembly Ritonavir/ Lopinavir (COVID-19) Boceprevir (HCV) Atazanavir (HIV) Inhibit protease
Inhibitors of viral release
- Although, on completion of virus replication and assembly, some drugs inhibit the last step, i.e., viral release from the host cell.
- Anti- Influenza or anti-COVID-19 drugs like oseltamivir block neuraminidase which is required to release a new virus. ❖ Besides the drugs that target viruses, some drugs have been developed that act as immunomodulators. For example, nitazoxanide interferes with host-regulated pathways of virus replication, amplification of type I interferon pathways, and cytoplasmic RNA sensing. ❖ Similarly, another drug, ivermectin , inhibits the nuclear import of host and viral proteins. Development of Antiviral Drugs and its Challenges Public health measures and vaccinations are an effective way to control viruses to a great extent. However, preventive measures are not always succeeded for numerous viral diseases. Antiviral drugs are necessary if the viral infection is life-threatening or causes serious illness. The first highly successful antiviral drug was acyclovir, developed during the 1970s, which was against HSV-1 and two and VZV. After that, antiviral drug discovery expanded markedly with HIV-AIDS epidemics. Meanwhile, different drugs were developed against the opportunists like HIV and CMV. With time, knowledge about viral genetics, molecular biology, enzymology, and protein structure led to the development modern and more effective approaches against viruses. Now, antiviral drugs are successful in saving lives and relieving suffering. One of the greatest achievements is that HIV is manageable for a lifetime as long as antiretroviral therapy is maintained in infected patients. In addition, an effective host response is required to recover any viral diseases. Inhibitors of viral release Oseltamivir (Influenza, COVID-19) and Zanamivir (Influenza) Block neuraminidase which is required for the release of a new virus.
Viruses have a short generation time (high number of replication cycles), which is one of the reasons for developing the resistant gene. The higher the replication magnitude, the higher the chances of mutation rate, and the more rapidly resistance can develop. A large virus population and drug-resistant mutants will be present among the array of genetic variants.
