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Sunday, December 27, 2009

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Friday, August 14, 2009

General Principles: Chemotherapy

General Principles of Chemotherapy
Many of the same basic principles apply to antimicrobial, antiparasitic and anticancer chemotherapy.
Selective Toxicity:
Selective toxicity refers to the ability of the drug to targets sites that are relative specific to the microorganism responsible for infection.
Sometimes these sites are unique to the microorganism or simply more essential to survival of the microorganism than to the host.
Examples of such specific or relatively specific sites include specific fungal or bacterial cell wall synthesizing enzymes, the bacterial ribosomal or the molecular machinery of viral replication.
Chambers, H.F., Hadley, W. K. and Jawetz, E. Introduction to Antimicrobial Agents in Basic and Clinical Pharmacology, (Katzung, B. G., ed) Appleton-Lange, 1998, p. 723.
Chemotherapeutic Drug Targets
Targets for Antimicrobial/Antiviral Drugs
Bacterial Cell Wall Synthesis Inhibitors
Agents that Increase Cell Membrane Permeability
Protein Synthesis Inhibitors: interfere with 30S or 50S bacterial ribosome function

Drugs that Bind to the 30S bacterial ribosomal subunit, leading to cell death
Agents that interfere with nucleic acid synthesis
Antimetabolites
Inhibitors of Viral Replication

Bacterial cell wall synthesis inhibitors:
penicillins
cephalosporins
cycloserine
vancomycin (Vancocin)
bacitracin
miconazole (Monistat)(imidazole antifungal)
ketoconazole (Nizoral)(imidazole antifungal)
clotrimazole (Mycelex)(imidazole antifungal)
Agents that increase cell membrane permeability
polymixins (detergent)
colistimethate (detergent)
nystatin (Mycostatin)(polyene antifungal)
amphotericin B (Fungizone, Amphotec)(polyene antifungal)

Protein synthesis inhibitors: interfere with 30S or 50S bacterial ribosome function.
Bacteriostatic
chloramphenicol (Chloromycetin)
tetracyclines
erythromycin estolate (Ilosone)
clindamycin (Cleocin)
Drugs that bind to the 30S bacterial ribosomal subunit, leading to cell death.
Bacteriocidal
Aminoglycosides (e.g.gentamicin (Garamycin), tobramycin (Nebcin))

Agents that interfere with nucleic acid synthesis
rifamycins (rifampin (Rimactane)): inhibits DNA-dependent RNA polymerase
quinolones: inhibit gyrase

Antimetabolites
sulfonamides
trimethoprim (generic)



Some Nucleic Acid Analogs (Antivirals): inhibitors of viral replication
zidovudine (Retrovir, AZT, azidothymidine)
ganciclovir (DHPG, Cytovene)
vidarabine (Vira-A)
acyclovir (Zovirax)
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1029-1030

Disease caused by bacteria and the the role of the host response in outcome of chemotherapeutic intervention.
Importance of Host Response
Host response as manifest in the inflammatory response is crucial for both the interruption resolution of the infection and for the basis of the infection's signs and symptoms.
The ability of effective antibiotic chemotherapy depends not only on appropriate selection of medication(s), dosage, and interval, but also on the host immune response.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1994, p. 592.
Disease Manifestations of Bacterial Disease
Disease manifestation of bacterial infection involves:
Colonization
Invasion: Bacterial invasion refers to the presence of bacteria in tissue sites normally sterile.
Examples:
Gram-negative rods in the blood stream result in septisemia and bacteremia without requiring endotoxin involvement
Pneumococcal pneumonia is due the growth of Streptococcus pneumoniae in the lung while bacterial endotoxins do not appear to play a role.
Disease that occurs after bactermia and invasion of the meninges by meningitis-producing bacteria (N. meningitidis, H. influenzae, E. coli, K1 and group B streptococci) is due to tissue destruction secondary to bacterial growth and host inflammation.
Toxin production and release
Toxin Elaboration
Clinicial manifestation of some bacterial infections are primarily due to toxin elaboration.
For example: Botulinum toxin: C. botulinum, Tetanus toxins: C. tetani, Diptheria toxin causes the disease due to infection with C. diphtheriae
Some specific aspects of bacterial disease are caused by elaborated toxins
Enterotoxins cause the diarrhea associated with E. coli, Salmonella, Shigella, Staphylococcus and V. cholerae.
Toxins involved in Toxic shock syndrome caused by Staphylococci, steptococci, P. aeruginosa and Bordatella: include: Toxic shock syndrome toxin (TSST), erythrogenic toxin, exotoxin A and pertussis toxin.
Staphylococcal enterotoxins, TSST-1 and streptococcal exotoxins have been classified as superantigens which are capable of inducing certain T cell proliferation without processing of the protein toxin by antigen-presenting cells.
This process involves, in part, the elaboration of IL-1 and TNF-alpha which may cause many clinical features seen in staphylococcal toxic shock syndrome, scarlet fever, streptococcal toxic shock syndrome.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 855.
Endotoxins (lipid A portion of gram-negative LPS) may cause many clinical presentations seen in gram-negative bacterial sepsis: Toxins involve include: IL-1 and TNF-alpha--
Clinical presentations include:fever, intravascular coagulation, shock, and muscle proteolysis.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 855.
Importance of Host Response
Host response as manifest in the inflammatory response is crucial for both interruption and infection resolution and the infection's signs and symptoms.
The ability of effective antibiotic chemotherapy depends not only on appropriate selection of medication(s), dosage, and interval, but also on the host immune response.
Pier, G.B. Molecular Mechanism of Bacterial Pathogenesis., In Harrison's Principles of Internal Medicine (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1994, p. 592.
Pharmacokinetic barriers that reduce the effectiveness of treatment
Introduction
Pharmacokinetic components include absorption, distribution, biotransformation (metabolism) and excretion.
Pharmacokinetic "profile" of antibacterial describes the drug concentration in tissues and serum as a function of time after administration.
The profile depends on the agent's absorption, distribution, biotransformation and excretion.
In therapeutics "trough" concentration (low) and "peak" concentrations (high) are important.
Pharmcokinetic information is used to establish dose and dosing intervals.
Absorption
Oral Therapy predominates for the following principal reasons: lower costs and fewer adverse effects
Another consideration, however, is that the oral route of administration is associated with a substantial range of bioavailabililty. For example, oral availability is only about 10% to 20% erythromycin estolate (Ilosone) and penicillin G, whereas bioavailability is about 100% for clindamycin (Cleocin), doxycycline (Vibramycin, Doryx) methonidazole, and trimethoprim (generic) sulfamethoxazole (Gantanol).
Bioavailability differences are NOT clinically important if the concentration of drug at the site of infection is sufficient to inhibit (bacteriostatic) or to kill (bacteriocidal).
Factors that can influence oral bioavailability include the presence of food in the digestive tract and drug interactions for example quinolones with metal cations.
Intramuscular
Administration by this route offers 100% bioavailable, but not widely used because of injection site pain can be usually ease of intravenous administration in the hospital inpatient setting.
Intravenous Administration:
Intravenous administration, however, would be appropriate if for example, oral agents proved ineffective, if there is a special concern about bioavailability, if larger doses are required relative to those typically obtained from oral dosing or because the bioavailability will be known to be 100%.
Distribution:
Antibacterial concentrations must exceed that required to inhibit bacterial growth (MIC).
Given that most infections are located outside the blood stream, the drug must distribute to those sites.
Drug concentration at most sites are similar to serum levels.
Some sites are "protected", however. these protected sites include the eye, prostate, and cardiac sites where there may be vegetative growth (e.g. around valves in a bacterial endocarditis setting)
High parentral doses may be required in these circumstances.
Poor efficacy may be related to adequate concentrations, but local unfavorable conditions.
For example, the activity of aminoglycosides is reduced in acidic pH, common at sites of infection. Biological constituants present in absesses may also inhibit the activity of antibacterials, thus requiring surgical dranage prior to efficacious therapy.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 860.

Mechanisms for chemotherapeutic drugs resistance
Bacterial resistance may occur because the drug does not reach its target site, drug is inactivated, or there is some sort of molecular alteration in the target itself, possibly due to mutation.
Resistance may occur because enzymes at or near the cell surface inactivate the antibiotic; the cell membrane is impermeable to the drug; there is an absence of aqueous channels (porins) through which the drug will reach the cell interior; there is a lack of a necessary transport system to support drug translocation; the transport mechanism is present but inoperative due to anaerobic metabolism; there are target site changes that results in reduced or absent antibacterial drug efficacy.
How Bacteria Acquire Resistance
Resistance may be acquired by vertical transfer, i.e. acquired by mutation and then passed to daughter cells
Mutations: Specific genetic mutations are the molecular basis for resistance to streptomycin (ribosomal mutation), to quinolones (DNA gyrase gene mutation) and to rifampin (Rimactane) (RNA polymerase gene mutation)
The mutation to rifampin (Rimactane) is an example of a single-step mutation: In this case E. coli or Staph. aureus exposure to rifampin results in highly resistant strain due to a point mutation in the RNA polymerase gene such that the polymerase protein no longer binds rifampin.
More usually, acquired by horizontal transfer of resistance factors from a donor cell, perhaps of a different species by transformation which involves the incorporation of DNA found free environment into the bacterial genome.
An example of this process is the basis of penicillin resistance in. pneumococci and Neisseria gonorrhoeae.
Penicillin-resistant pneumococci produce different PBPs (penicillin-binding proteins). These different PBPs exhibit relatively low affinity for penicillin compared to wild type pneumococci.
These different PBPs arise from integration of foreign DNA which were most likely from a closely related streptococcal strain into the PBP gene by a process of homologous recombination.
Transduction-based resistance occurs when a bacteriophage which includes bacterial DNA in its protein coat infects the bacteria. This bacterial DNA may contain a gene confiring resistance to antibacterial drugs.
Examples of this process:
Staphylococcus aureus strain resistance development to penicillin may occur by transduction (Some bacteriophages carry plasmids [extrachromosomal self-replicating DNA] that code for penicillinase
Other phages can transfer genes which confer resistance to tetracycline (Achromycin), erythromycin estolate (Ilosone), and chloramphenicol (Chloromycetin).
Conjugation is another important mechanism for single and multi-drug resistance development. In conjugation direct passage of resistance-confering DNA between bacteria proceeds by way of a bridge
The genetic material transfer in conjugation requires two elements: an R-determinant plasmid which codes for the resistance and a resistance-transfer factor (RTF) plasmid which contains the genes necessary for the bacterial conjugation process. Occasionally two plasmids join to form a complete R factor
Some genes that are responsible for resistance are located on transposons which can move from location to location within plasmid and bacterial genomes.
Conjugation mediated resistance is particularly important in gram-negative bacilli.
Enterococci may contain plasmids that spread resistance among gram-positive organisms
Vancomycin (Vancocin) resistance in enterococcal strains appears to occur as a result of the conjugation mechanism.
Conjugation is not a high efficiency mechanism for resistance development. Unfortunately, antibiotic use provides selection pressures which facilitate the elaboration of resistance bacteria. Furthermore, enteric bacteria carrying plasmids for multidrug resistance is now a worldwide, serious concern.
Resistance acquired by horizontal transfer can disseminate rapidly through the bacterial population by clonal spread as well as by continuing genetic material exchange between cells of the same or different susceptible strains.
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1031.
Microbial Resistance and Specific Drugs
Resistance: ß-Lactams
Most common among several mechanisms by which bacteria develop resistance to ß-Lactam antibiotics is by elaboration of the enzyme ß-lactamase, which hydrolyzes the ß-lactam ring.
Beta-lactamase genes may be found in both gram-positive and gram-negative bacteria. Resistance may be reduced by agents which bind to some beta-lactamases. Examples of these drugs include clavulanic acid and sulbactam.
Another mechanism by which bacteria may develop resistance to beta-lactam antibiotics is by changes in penicillin-binding proteins (PBPs). These changes may occur either by mutation of existing PBP genes or more often by the acquisition of new PBP genes. For the latter case, unimportant example is staphylococcal resistance to methicillin (Staphcillin).
In addition to mutation of existing PBP genes, bacterial also acquiring new "pieces or segments" of PBP genes. This process appears important in resistance development for certain pneumococcal, gonococcal, and meningococcal strains)
Yet another mechanism is observed in gram-negative bacteria and follows from alteration of genes that code for certain outer membrane proteins (porins). The expression of altered porins reduce membrane permeability to penicillins. This process appears important in cephalosporin-resistance of Enterbacteriaceae and that of Pseudomonas species to ureidopenicillins.
Multiple resistance mechanisms can exist within the same bacterial cell.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 859.
Chloramphenicol (Chloromycetin) resistance:
This resistance occurs because of the formation of an acetylated chloramphenicol (Chloromycetin) derivative which is not biologically active.
A plasmid-encoded enzyme, chloramphenicol acetyltransferase catalyzes the acetyl group transfer which would otherwise not occur
Vancomycin (Vancocin) resistance is an important problem in antibiotic chemotherapy. For example, vancomycin (Vancocin)-resistant enterococci strains are worldwide.
The resistance mechanism involves transfer between cells and is plasmid mediated.
The specific alteration is a change in the peptidoglycan stem peptide which causes a loss of the vancomycin (Vancocin) binding target.
Often significant vancomycin (Vancocin) resistance is observed with enterococci strains all the same time most staphylococci aureus and staphylococci epidermis remain vancomycin (Vancocin) sensitive.
Resistance to tetracyclines:
The most common mechanism for gram-negative bacterial resistance follows from insertion of a plasmid-encoded active-efflux pump which translocates tetracycline (Achromycin) out of the cell.
For gram-positive bacteria, resistance may follow from the above mechanism (active efflux) or is a result of changes in the tetracycline ribosomal target site.
Aminoglycoside resistance:
The most common resistance mechanism is antibiotic inactivation by enzyme-mediated covalent bond modification. Modifications occur as a result of acetyl, adenyl or phosphate group transfer.
Enzymes which catalyze these group transfers and confer antibody resistance are plasmid localized.
As a consequence of these molecular modifications, the modified antibiotic becomes less active secondary to reduced transport and attenuated ribosomal target site binding.
Aminoglycoside-modifying enzymes occur in both gram-negative and gram-positive bacteria.
Mupirocin resistance: Resistance to this topical drug is due to alteration in the target site which is an isoleucine tRNA synthetase enzyme. Following modification, the enzyme no longer binds mupirocin (Bactriban)
Macrolides and Licosamides resistance. Antibiotic from these categories target gram-positive bacteria.
Resistance to these antibiotics result from plasmid-mediated ribosomal RNA methylation that interferes with antibiotic binding.
Specific antibiotic susceptible to this type of resistance include erythromycin, newer macrolides and clindamycin (Cleocin).
Quinolone-resistance:
Quinone resistance including resistance to newer fluoroquinolones has developed quickly in Staphylococcus and Pseudomonas strains.
The resistance mechanism involves a mutation in the drug target site, which is DNA gyrases. The mediated DNA gyrases are not susceptible to inhibition by quinolones.
Other resistance mechanisms involve in gram-negative strains, mutations in porins that cause the bacterial membrane to lose its permeability to the drug.in gram-positive organisms, resistance follows the development of the capability that enables the cell to actively pump out the drug from the cell.
Rifampin (Rimactane) resistance can rapidly develop as a result of target site mutations. The target in this case is RNA polymerase. Following mutation of the polymerase, rifampin (Rimactane) no longer binds.
Rapid strain resistance development has become a major limiting factor in rifampin (Rimactane) use in management of susceptible staphylococci thus requiring rifampin (Rimactane) to be combined with another antistaphylococcal drug.
Multiple Antibiotic Resistance Summary
It is increasingly common for one bacterium to be resistant to several antibacterial drugs.
Mechanisms:
Acquisition of multiple, unrelated resistance genes (several steps required)
Mutation in a single gene which results in resistance to unrelated drugs. (single step)
Bacteria resistant due to acquisition of new genes:
hospital-associated gram-negative bacteria
enterococci
staphylococci
community-acquired salmonellae strains
gonococci
pneumococci.
Single gene mutations: usually affecting porins of gram-negative bacteria involve:
ß-lactams
quinolones
tetracycline
chloramphenicol (Chloromycetin)
trimethoprim (generic)
Strains resistant to all known antibacterial drugs have been identified.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 859.
Clinical indications and rationale for the use of multiple drugs at the same time (combination chemotherapy)
Single Agent Chemotherapy
Single most specific drug is preferable if the infecting bacteria has been identified.
Administration of a single drug with a narrow spectrum of action is desirable because (a) alteration of normal flora is minimized (which in turn reduces the likelihood of overgrowth of resistant nosocomial bacteria (e.g. Candida albicans, enterococci, Clostridium difficile), (b) reduces toxicity which may be associated with multiple drug regimens and (c) reduces cost.
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 862.
Combination Chemotherapy
Combination chemotherapy may be warrented to:
Decrease the likelihood of emergence of resistant mutants. A single agent will be effective against sensitive organisms, but not against those that have developed a mutated "target" site, which is no longer susceptible or has diminished susceptibility to the drug. In this case the single drug will select out the mutant, resistant strain. This effect is more likely when the concentration of the antibacterial agent approximates the MIC (minimum inhibitory concentration).
Examples:
rifampin (Rimactane) : staphylococci
ciprofloxacin (Cipro): staphylococci and Pseudomonas.
imipenem: Pseudomonas
aminoglycosides: staphylococci
A second agent, which acts by a different mechanism, may prevent the emergence of the resistant strain (e.g. impenem + aminoglycoside for systemic Pseudomonas).
To take advantage of additive/synergistic action against some bacteria. Synergistic or additive activity occurs if the MIC or MBC of each agent is lowered in the presence of the other. Accordingly, each drug is more efficacious when combined with the other. (1) Certain ß-lactam-aminoglycoside combinations are effective against enterococci, viridans streptococci, and P. aeruginosa. (2) Combination of trimethoprim-sulfamethoxazole (Bactrim) is effective against many enteric gram negative bacteria. (3) Most other combinations are not clinically surperior compared to administration of the more efficacious single drug component.
Some combinations are less effective than a single agent: penicillin plus tetracycline (Achromycin) against pneumonococci.
To provide therapy when multiple pathogens may be or are known to be present . If a mixture of pathogens is thought to be present and/or the patient is critically ill, combination therapy may be warrented. [Multiple Pathogens may be present in intra-abdominal or brain abscesses and limb infection in diabetic patients; in critical illness fevers in neutropenic patients, acute aspiration pneumonia (oral flora) by hospitalized patients, septic shock or sepsis syndrome. However, monotherapy should be started if a single infecting bacterium that can be appropriately treated with a single drug has been identified
Archer,G.L. and Polk, R.E. Treatment and Prophylaxis of Bacterial Infections, In Harrison's Principles of Internal Medicine 14th edition, (Isselbacher, K.J., Braunwald, E., Wilson, J.D., Martin, J.B., Fauci, A.S. and Kasper, D.L., eds) McGraw-Hill, Inc (Health Professions Division), 1998, p. 862.


Rationale for chemoprophylaxis
Chemoprophylaxis
A manifestation of antibiotic misuse is that 30% to 50% of the time, the antibiotic is prescribed to prevent rather treat an infection.
Prophylaxis is more likely to be effective if a single, effective, nontoxic drug is used to prevent infection by a specific organism or to eliminate a recently established infection.
Examples of effective chemoprophylaxis:
Penicillin G prevents infection by group-A streptococci.
Intermittent use of trimethoprim (generic) sulfamethoxazole (Gantanol) prevents recurrent urinary tract infections
Prevention of endocarditis in patients with valvular heart lesions who are to undergo a surgical procedure.
The most extensive use of chemoprophylaxis is prevention of wound infections following surgery.
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1049-1050
Appropriate and Inappropriate Uses of Chemotherapy
Appropriate Use
Before the organism is identified: either combination therapy or a single broad spectrum agent may be used.
After the organism is identified, a low-toxicity regimen with a narrow-spectrum drug is indicated.
Selection of the drug should be goverened by its selectivity for the most likely involved bacteria and its toxicity.
First decide if an antibiotic is indicated since antibiotics may be toxic. Inappropriate use may hinder diagnosis, and can result in development of resistant bacterial strains
Some Clinical Issues
Optimal empirical treatment requires knowledge of the antibiotic sensitivity of the organisms which is most likely causing the infection.
Assessment with Gram's stain and other tests must be used to narrow the list of pathogens.
In life threatening situations, the selection of a single narrow-spectrum agent may not be possible and broad coverage would be indicated until a definitive identification is possible.
Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacologial Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1054-1055
Inappropriate Uses
Treatment of Untreatable Infections:
The infection is viral.
Antimicrobial treatment of measles, mumps and 90% of upper respiratory infections are inappropriate.
Treatment of fever of unknown origin:
Antimicrobials are not antipyretic agents.
Pyrexia of short duration, without localizaing signs, is most likely due to viral infection.
Three infections may be associated with prolonged fever:
tuberculosis
hidden intra-abdominal abscesses
infective endocarditis.
other causes: cancer metabolic disorders hepatitis atypical rheumatoid arthritis
Improper dosage:
Some drugs, such as the aminoglycosides, are frequently administered at subtherapeutic dosages because of concern about toxicity.
Clinical treatment failure and selection of resistant strains may result.
Inappropriate Dependence on Chemotherapy Alone:
Some disorders require both chemotherapy and a surgical procedure, especially if significant amount of necrotic tissue is present.
Example : Pneumonia in a patient with empyema (accumulation of pus) may be effectively manage following drainage
Lack of Adquate Bacteriological Information:
About one-half of antimicrobial therapy is given to hospitalized patients without support from microbiological data. [clinical judgment alone];
Antimicrobial therapy must be individualized, not administered based on prescribing habit alone.

Chambers, H.F and Sande, M.A. Antimicrobial Agents in,In, Goodman and Gillman's The Pharmacological Basis of Therapeutics,(Hardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc.,1996, pp.1054-1055

Wednesday, July 8, 2009

Chemical aspects of drugs

Drug shape
The shape of the drug is an important factor in defining the nature of the drug-receptor interaction. The three-dimensional shape of the drug is thought to interact with a complementary structural binding region of the receptor, typically a protein. The specific nature of the interaction defines whether the drug acts as an agonist promoting a change in cellular function or as an antagonist which blocks the receptor usually resulting in no direct biological effect.
For example, let's consider acetylcholine or a synthetic analogue bethanechol (Urecholine). Interaction of these molecules with receptor (nicotinic or muscarinic cholinergic receptor) causes a physiological response -- a decrease in heartbreak for instance. By contrast, a muscarinic antagonist such as atropine may bind even more tightly than acetylcholine to muscarinic receptor but causes no direct effect. However, following administration of antagonist a biological response may be observed as a result of receptor blockade.
A clinical example would be bradycardia following acute myocardial infarction. Bradycardia in this context might be due to excessive parasympathetic (cholinergic) tone and might cause unacceptably low cardiac output or predispose tomore serious arrhythmias. Administration of atropine, by blocking the muscarinic receptor blunts the action of acetylcholine and accordingly may reverse bradycardia.
Now let's consider the specific example,acetylcholine, as the 2D planar structure:
On the left side of the molecule note the quaternary (always positively charged) Nitrogen, which is part of the choline component of acetylcholine. The synthesis of acetylcholine proceeds by combination of choline and acetate (as Acetyl-CoA)-see below
Now let's examine the 3-D structure of choline (pressing and holding down your left mouse button allows you to rotate the choline 3-D interactive structure). By convention, nitrogen atoms are blue, oxygen red and carbon gray.
Similarly we can examine interactively the 3-D structure of acetylcholine:

Note above the presence of an "ester" linkage [O in red] between the choline moiety and the seal group.
This ester bond is susceptible to hydrolysis, i.e. breakage which may be catalyzed by esterases (acetylcholinesterase is an example).
Acetycholine:

Although acetylcholine is depicted as a "static" molecule in terms of internal rotation,, acetylcholine and many other drugs exhibit free rotation around internal bonds.
For acetylcholine,tau1, tau2, tau3, represent torsion angles and refer to the degree of twist around these bonds of free rotation
Specific additional analysis is required to determine which three-dimensional form of acetylcholine appears to be preferred for binding to the cholinergic receptor. The configuration of acetylcholine and solution is quite different than the configuration when bound to the nicotinic cholinergic receptor (using two-dimensional NMR to estimate bond angles)

Above figures adapted from Principles of Drug Action: The Basis of Pharmacology, Third Edition, edited by William . B. Pratt and Palmer Taylor, Churchill Livingston, New York, 1990. pp 20-23. Above Acetylcholine conformation figure -- original work: Behling, RW, Yamane T, Gavon G, Jelinski LW: Conformation of Acetylcholine Bound to the Nicotinic Acetylcholine Receptor. Proc Natl Acad Sci USA 85:6721, 1988.
Some short-acting pharmacological agents are in fact short-acting because they are rapidly hydrolyzed at an ester linkage.
Ester-type local anesthetics
Esmolol (Brevibloc)
Remifentanil (Ultiva).
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The biological action acetylcholine is terminated by hydrolysis, catalyzed by the enzyme acetylcholinesterase: The overall reaction is shown below --
Acetylcholinesterase itself is a large, complex protein which has its primary catalytic function the extremely rapid hydrolysis of the neurotransmitter acetylcholine.
Acetylcholinesterase
The image below illustrates the relationship between the very small molecule, acetylcholine, and its specific interaction within the very large molecule, acetylcholinesterase.
This image illustrates how the neurotransmitter acetylcholine represented above the in ball-and-stick form is recognized by specific amino acids within acetylcholinesterase's active site.
The positive charge of acetylcholine (due to the permanently positive quaternary nitrogen) interacts with tryptophan-84 (Trp-84) and phenylalanine-330 (Phe-330), through cationic (+ charged)- π-electron interactions}
This part of the acetylcholinesterase molecule is referred to as the "aromatic gorge"
The negatively charged amino acid, glutamatic acid-199 (Glu-199) is thought to interact with acetylcholine through ionic-type interactions

Thursday, May 28, 2009

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Saturday, May 2, 2009

The Importance of Microbiology in the Contamination Control Plan for Aseptic, Terminally Sterilized and Non-sterile Manufacturing

Introduction
The development of a contamination control program is critical to the effort to get a new facility qualified, and to maintain the facility in a state of control once qualified. The design and successful execution of a contamination control program requires a plan. The creation of a specific document allows the company philosophy, goals, and expectations to be formalized and agreed to by all parties. It also provides the goals and metrics by which the state of control for the facility can be measured in the annual review. The business reasons for this are obvious in terms of reduced regulatory risk and reduction of rejected/recalled batches (Lowry 2001).
This plan is important no matter what type of facility is being developed. Although it is most frequently used in the Quality plan for commissioning an aseptic facility, this is also important and should be used for commissioning and controlling facilities using terminal sterilization, and for non-sterile manufacturing facilities.
Why be concerned with contamination control in a nonsterile manufacturing facility? In many ways contamination control is more of a concern in a non-sterile facility than in sterile product production facilities. The sterile production facility knows there is a problem with contamination and cross-contamination of batches, the non-sterile facility has a great temptation to belief they are not touched by these issues. This can lead to an extremely cavalier attitude about contamination control by the operators and management. The non-sterile manufacturer is responsible for all aspects of his product, including any objectionable organisms present (Sutton, 2006) as described in a recent newsletter (PMF Newsletter v12 n7).
The API manufacturer is also concerned with contamination control. The FDA has explicit instruction on this score (FDA 1998) out of CBER. The EMEA guidance on API manufacture also includes guidance on control of bioburden and cross-contamination of batches (EMEA 2000).
This essay will not be able to provide more than an overview of issues in the space available this month. However, it is hoped that the need for an adequate contamination control plan for a facility will be made clear, and the beginnings of the content of such a plan explained. The interested reader is referred to the articles listed in the “References” and the “Further Readings” sections.
Scope
The Contamination Control Plan should be developed as part of the facility commissioning effort. As such, there will be four distinct phases of the facility operations that will need to be addressed:
Commissioning and initial start-up
Ongoing Operations
Shut-down for regular maintenance
Start-up after scheduled shut-down.
These phases will not have the same level of contamination control. In fact, the third and fourth phases may well have different levels of control to be addressed. A good plan will discuss the concerns specific to each of these phases.
This program, and the protocol governing the program, are essential documents useful in documenting the rationale and methods used to accomplish three tasks:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
As the SME (Subject Matter Expert) in microbiology, we will be most heavily involved in the first of these three tasks, minimizing bioburden. However, all three will be discussed (at least briefly) in this essay for context.
Minimizing Bioburden

Validated methods
All measures of bioburden in a facility will be indirect. We cannot count bacterial cells on a surface or in the air. We must transfer the microorganisms to an agar plate (or some other mechanism) and count colony forming units. If we make the assumption that the transfer of microorganisms from the air or from a surface to agar is consistent, then we can use these numbers to estimate trends over time. This assumes that the nutrient agar is capable of growing the microorganisms to visible colonies. As residual disinfectant on a surface may impede the growth of microorganisms, neutralizers are frequently incorporated into the growth media (Dey-Engley agar, MCTA, etc.). All sampling methods must be validated for the conditions of use.
The facility should be disinfected regularly using validated sanitizers and sporicides. The contamination control plan should describe the methods for testing and rationale for acceptance of materials to be used in the ongoing program of disinfection. The plan should ideally describe the in vitro or laboratory tests to evaluate the sanitizers, including the identification of the most resistant microorganisms found in the facility as well as the most difficult-to-disinfect materials in the facility. This is also where the method for on-going evaluation of the sanitizers based on environmental monitoring data will be recorded. The choice of disinfection regimens should be reevaluated annually, and the contamination control plan should describe how this evaluation will occur.
Know the enemy
A successful contamination control program is geared to providing the most useful information on the microorganisms present while at the same time showing some fiscal responsibility. The FDA aseptic processing guidance document recommends genetic identification of all organisms isolated from the manufacturing environment on a regular basis. (FDA, 2004) This is a laudable goal, but few of us have anything near the required budget to accomplish this task, and in all honesty it is reasonable to wonder if it is really necessary. The numbers of CFU from validated sites (viable air and surface, non-viable) is sufficient to provide a measure of the state of control of the facility. However, periodic cataloging of the resident microflora will provide you with a good check on the continued effectiveness of the disinfectants in use. Shifts of bioburden to spore forming microorganisms will be strong evidence of the need for use of a sporicidal agent. Occasionally, this effort will also pick up shifts among non-spore-forming organisms – this is not due to “resistance” but rather ecological shifts towards species more naturally resistant to the disinfectant in use.
Control incoming bioburden
The first step in any control program is to control contamination at the very beginning of the process. This includes raw materials (excipients, API, water, etc) and the primary containers. All materials should be tested for incoming bioburden against documented acceptance criteria. Part of the incoming bioburden will also be any water used as an excipient to the process. A good guide for the water bioburden is the EMEA guidance on the subject (EMEA 2002).
Appropriate gowning
The gowning methods and materials are of critical importance to minimization of contamination. Although most attention is placed on aseptic gowning procedures, the appropriate use of gowning precautions will be a great boon to most non-sterile manufacturing facilities as well. All personnel should be well-trained in appropriate gowning practice and behavior. The contamination control plan should describe the rationale for the level of gowning chosen, the frequency of gown cleaning, behavior and the acceptable gown materials for the type of manufacturing process.
Training
Operator training is critical to contamination control. No supervisor can be present at all locations at all times. Each operator must be aware of his or her role in contamination control and how to minimize the risk to batch integrity. The PDA has published a technical report that speaks to some of these training requirements from the microbiological perspective (PDA 2001).
Controlled Environments
Control and monitoring of the environment is another critical element of the contamination control plan. Large portions of this can be addressed by the corporate Environmental Monitoring Master Plan (which provides rationale and consistency for a single EM philosophy across the different facilities of the corporation) or the site Environmental Master Plan (which provides consistency and detailed instruction for the various manufacturing buildings at a given site). However, the Contamination Control Plan should cite the relevant documents and their role in contamination control. Those interested in more on environmental monitoring should refer to the PDA’s treatment of the subject for a good overview (PDA 2001).
The appropriate Environmental Monitoring (EM) plan for non-sterile manufactures and for API manufacturers is not well-defined from a regulatory sense. There are no strong recommendations such as those seen for the environmental monitoring of aseptic facilities; however the absence of regulatory guidance is not the same thing as the absence of need for the activity. EM is useful for determining the state of control of the facility (not so much, perhaps an indicator of the finished product quality) and so is an important part of the monitoring program for all manufacturers.
Well-defined and Understood Manufacturing Processes
The manufacturing process should be evaluated for its potential to limit or eliminate bioburden. The two common methods for performing this is either a HACCP-type (Jahnke and Kuhn 2003) or a FMEA approach. The use of organic solvents, heat, or other inhospitable activities can greatly reduce bioburden of a process. The contribution of compression (and associated shear), for example, should be evaluated for a potential reduction in risk of excessive microbial contamination (Blair 1991). The contribution of the finished product water activity should also contribute to this analysis (USP 2007).
Of particular importance in this evaluation for the potential for microbial contamination of the process are cleaning steps, equipment hold times, HVAC, control level of environments for critical tasks, open-system vs closed-system operations, and bioburden monitoring (among others specific to your process). As an example of the importance of the bioburden control point issue, there is a strong regulatory expectation in Europe that products sterilized by filtration should have a pre-filtration bioburden of not more than 10 CFU/100 mL immediately before the sterilizing filter.
Finally the Contamination Control Plan should cite the need clear SOPs on all aspects of manufacturing, monitoring and control. These SOPs are critical for training, documentation and batch release.
Minimization of Batch Residual Cross-over Contamination
The contamination control plan should also address the potential for a batch to be contaminated by material from the previous batch manufactured using that equipment. Obviously, the contamination control plan should describe the methods by which this likelihood is minimized.
The concern over batch residual cross-over is most relevant when there is more than one product manufactured at a site. This concern has little to do with the sterility of the finished product, and is relevant to sterile and non-sterile manufacture alike.
Minimization of Cleaning Material Residual Contamination
Validation of cleaning procedures is essential to demonstrate not only that the cleaning procedure effectively cleans and sanitizes the manufacturing equipment, but also that residual cleaning material is removed to prevent contamination of the next batch manufactured.
Conclusions
The Contamination Control Plan is an important document designed to formalize the rationale, methods and validation of contamination control procedures in a manufacturing facility. This plan is a valuable tool for pharmaceutical, medical device and personal product manufactures and should be written to address all phases of the facilities life cycle. The Contamination Control Plan should specifically address:
Minimizing the bioburden throughout the manufacturing processes
Minimizing the level of batch residual cross-over contamination
Minimizing the level of cleaning material residual contamination
The microbiologist, as SME, has a critical role to play in the first of these three primary goals, and this essay has therefore been directed at that first topic. Minimization of bioburden in the manufacturing process occurs through (but is not limited to):
Minimizing bioburden in the process
Control incoming bioburden
Appropriate Gowning
Controlled Environments
Well-defined Standard Operating Procedures; and
Well-defined and understood manufacturing processes.

Microbial Limits Tests: The Difference Between “Absence of Objectionable Microorganisms” and “Absence of Specified Microorganisms”

We have to note from the outset that USP and FDA frequently are interested in the same thing. From the vantage point of USP, there is a need to have a test for sterility, for antimicrobial efficacy, for Antibiotic/ Vitamin Potency, for Bacterial Endotoxin, for Microbial Limits etc. The need for these tests is not driven by any concern over “Good Manufacturing Process” (GMP). It is governed by the USP monographs found in the National Formulary (NF). If there is a monograph that requires a test for antimicrobial efficacy, then chapter <51> Antimicrobial Effectiveness Test” is the referee test used to demonstrate that characteristic.
FDA has similar, but separate concerns. Where the requirements are identical, the referee chapters in USP (those numbered under <1000>) are enforced. However, there are situations where the FDA’s concerns are not covered by a USP referee test method. One such situation is with the CFR requirement that medicines be “free of objectionable microorganisms.” 21CFR 211.113 under the section “Control of microbiological contamination. (a)” states “Appropriate written procedures, designed to prevent objectionable microorganisms on drug products not required to be sterile, shall be established and followed.” This is reinforced by 21 CFR 211.165 which states “Testing and release for distribution... (b) There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms.”
So, here we have a problem. The USP monograph for a product (as provided in the current National Formulary) may require “Absence of Pseudomonas aeruginosa.” There is a test in the Microbial Limits chapter to demonstrate the absence of Pseudomonas aeruginosa. However, although this test may be required to demonstrate compliance with the monograph requires as laid out in NF it does not meet the FDA concern that any organism in the final product be acceptable to the product and the target population (i.e. are not “objectionable”).
The FDA Concern
FDA will enforce the GMP requirement that if your product approval to market submission contained a statement that you would test the finished product by the Microbial Limits Tests that in fact you must do that. This is purely a GMP concern. However, the Agency has been absolutely clear on the concern over objectionable microorganisms in the product, and that fact that testing to the USP chapter might be necessary, but it is not sufficient to demonstrate microbial quality. In fact, in the 1993 instructional guide for inspections of QC Microbiology Labs (1) the FDA states:
“For a variety of reasons, we have seen a number of problems associated with the microbiological contamination of topical drug products, nasal solutions and inhalation products. The USP Microbiological Attributes Chapter <1111> provides little specific guidance other than "The significance of microorganisms in nonsterile pharmaceutical products should be evaluated in terms of the use of the product, the nature of the product, and the potential hazard to the user." The USP recommends that certain categories be routinely tested for total counts and specified indicator microbial contaminants. For example natural plant, animal and some mineral products for Salmonella, oral liquids for E. Coli [sic], topicals for P. aeruginosa and S. Aureus [sic], and articles intended for rectal, urethral, or vaginal administration for yeasts and molds. A number of specific monographs also include definitive microbial limits.
As a general guide for acceptable levels and types of microbiological contamination in products, Dr. Dunnigan of the Bureau of Medicine of the FDA commented on the health hazard. In 1970, he said that topical preparations contaminated with gram negative organisms are a probable moderate to serious health hazard. Through the literature and through our investigations, it has been shown that a variety of infections have been traced to the gram negative contamination of topical products. The classical example being the Pseudomonas cepacia contamination of Povidone Iodine products reported by a hospital in Massachusetts several years ago.
Therefore, each company is expected to develop microbial specifications for their nonsterile products. Likewise, the USP Microbial Limits Chapter <61> provides methodology for selected indicator organisms, but not all objectionable organisms. For example, it is widely recognized that Pseudomonas cepacia is objectionable if found in a topical product or nasal solution in high numbers; yet, there are no test methods provided in the USP that will enable the identification of the presence of this microorganism.
A relevant example of this problem is the recall of Metaproterenol Sulfate Inhalation Solution. The USP XXII monograph requires no microbial testing for this product. The agency classified this as a Class I recall because the product was contaminated with Pseudomonas gladioli/cepacia. The health hazard evaluation commented that the risk of pulmonary infection is especially serious and potentially life-threatening to patients with chronic obstructive airway disease, cystic fibrosis, and immuno- compromised patients. Additionally, these organisms would not have been identified by testing procedures delineated in the general Microbial Limits section of the Compendia. . . .
Microbiological testing may include an identification of colonies found during the Total Aerobic Plate Count test. Again, the identification should not merely be limited to the USP indicator organisms.
The importance of identifying all isolates from either or both Total Plate Count testing and enrichment testing will depend upon the product and its intended use. Obviously, if an oral solid dosage form such as a tablet is tested, it may be acceptable to identify isolates when testing shows high levels. However, for other products such as topicals, inhalants or nasal solutions where there is a major concern for microbiological contamination, isolates from plate counts, as well as enrichment testing, should be identified.”
Why is this a concern? To understand this we have to go back to the 1970’s. USP had a test for the “Bacteriological Examination of Gelatin” as early as 1942 (2). However, most non-sterile medications in the US were not required to assay for microbiological quality attributes until the introduction of the Microbial Limits Tests in 1970 (3). In the late 1960’s several outbreaks of disease were traced back to pathogencontaminated medications, and this prompted increased attention to the microbial content of non-sterile drugs (4). Later in the 1980’s there was a series of articles appearing in the literature describing contamination by P. cepacia (currently Burkholderia cepacia) (5, 6) and its survival in disinfectants(7 – 11). This lead to the addition of requirements in the 21 CFR to ensure that there are not objectionable organisms in product released to market (see above). Add to this the knowledge that the USP “Absence of Pseudomonas aeruginosa” assay will not identify presence of B. cepacia (as discussed).
The USP Concern
The USP is on record as early as 1982 verifying that the demonstration of “absence of objectionable microorganisms” is not the intent of the chapter. In a one page Stimuli to the Revision Process the microbiology committee of the time states:
“The tests described in the Microbial Limits Tests <61> were not designed to be all-inclusive, i.e., to detect all potential pathogens. To accomplish this, an extensive text on laboratory detection of microorganisms would be required. The procedures in USP were designed to detect the presence of specific “index” or “indicator” organisms. Nevertheless, the present chapter does not preclude the detection of Ps. Cepacia – the organism requires subsequent differentiation. The chapter does not provide specific methods for this, nor does it provide procedures for detecting thousands of other potentially pathogenic organisms. Individual monographs include requirements for limits on total aerobic counts and/or absence of one or more of the four selected “indicator” organisms. The chapter on Microbial Limits Tests provides methods to assure that one may test for those microbial requirements in the individual monographs...
In conclusion, the Microbial Attributes and Microbial Limit Tests chapters accomplish their intent. If a manufacturer needs particular tests for any specific organisms that are potential problems in a process or a final product, the quality control microbiologist can provide specific detection procedures. Many such procedures are published in several laboratory texts on microbiology.”
Conclusions
On the question of the microbial quality of non-sterile pharmaceuticals, the USP and the FDA are in agreement – the product must be safe for use. The NF monograph requirements for absence of specific organisms is a minimal requirement, and should not be taken as proof that the product is suitable for sale from a microbiological perspective.
The manufacturer is responsible for the quality and safety of the product marketed, and it is the clear expectation of FDA (as described in CFR) that this will include a determination of the microbial safety – i.e. the “absence of objectionable microorganisms” from the product. These positions have been publicly stated for decades and should not come as a surprise to anyone. The harmonized microbial limits tests only address the “absence of specified microorganisms” and leave the determination of the “absence of objectionable microorganisms” in the capable hands of each company’s appropriately educated and well-trained microbiology group.

The Harmonization of the Microbial Limits Test - Absence of Specified Organisms

The last issue of the PMF Newsletter (vol 12, no. 3) contained a review of the harmonization status of the Microbial Limits Tests – Enumeration. In addition, the article provided a brief overview of the compendial harmonization process as agreed to by the Pharmacopeial Discussion Group (PDG). In this article, I would like to focus on the other side of the Microbial Limits Tests – the “Absence of Specified Microorganisms” component.
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
There is a significant controversy in the United States over the intent of this evaluation. The FDA is bound by the concern expressed in the Code of Federal Regulations (21CFR 211.113 and 21CFR 211.165) relating to the importance of “objectionable microorganisms.” This is not the concern of the compendial chapters. The controversy is worthy of discussion, but not the topic of this review – it will be discussed in length in the final of three articles on the harmonization of the microbial limits tests to be published in next month’s newsletter.
What follows is a tabular presentation of the existing “Microbial Limits – Absence of Specified Microorganisms” tests from the current USP and Pharm Eur, as well as the draft harmonized document (the finalized document is extremely close to this version, but not release to the industry). It is provided as an aid to evaluation, and may assist in determining whether revalidation of method suitability studies is needed. It should be noted that this harmonization draft represents a true compromise by all parties, with (at least in the author’s opinion) significant changes from the current USP, Pharm Eur and JP chapters.

The Harmonization of the Microbial Limits Test - Enumeration

The USP and the European Pharmacopoeia (EP, Pharm Eur) Microbial Limits Tests are in the final stages of harmonization. They were signed off to Stage 6A at the November, 2005 meeting of the Pharmacopeial Discussion Group (PDG) held in Chicago, IL USA (USP 2006a). However, the signed-off versions have yet to be published. This makes the description of the test a bit difficult, as the current tests will be disappearing, and the final, harmonized test is not yet public knowledge. However, we do know that the harmonized tests do not differ greatly from the drafts published in 2003 (USP 2003a, USP 2003b, USP 2003c), and so we will use those drafts as the description of the finalized test.
The Microbial Limits Tests are actually two chapters in the current USP: Current USP <61> Microbial Limits Tests (USP 2006b) and <1111> Microbiological Attributes of Nonsterile Pharmaceutical Products (USP 2006c). This will be modified in the harmonized version to mirror the European format:
USP
EP
<61> Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
2.6.12 Microbiological Examination Of Nonsterile Products: Microbial Enumeration Tests
<62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
2.6.13 Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms
<1111> Microbiological Quality of Nonsterile Pharmaceutical Products
5.1.4 Microbiological Quality of Nonsterile Pharmaceutical Products
Table 1: Harmonized Chapter Numbering Scheme
This review will only address the microbial enumeration portions of the harmonization effort – that which will become USP chapter <61> and Pharm. Eur. chapter 2.6.12.
The microbial enumeration test is a basic, simple design to count the number of CFU in a nonsterile product or raw material. The preferred method is to put the material into solution and then plate aliquots to determine the CFU/gram (or mL) of initial material. If the product cannot be put into solution, there are provisions to use the Most Probable Number method (MPN – see FDA BAM website). The method of plating can be either pour plate, spread plate or the filtration of material and then placing the membrane filter on the surface of an agar plate. The membrane filtration method should only be used when there are few expected colony forming units in the material to be tested as it is a good method to test a large volume of liquid, but can only count up to approximately 100 CFU/membrane.
The harmonized method provides a great deal more detail than any of the current pharmacopeial methods in terms of demonstration of method suitability (validation of the method) and in terms of media growth promotion.
The demonstration of method suitability should be performed using the challenge organisms listed (see Table 2 below) in accordance with the recommendations found in USP chapter <1227> (USP 2006d). Growth promotion is an area of some ambiguity in the compendial text. Although media growth promotion is not described in the tests, demonstration of media suitability is required, and the draft USP Chapter <1117> (USP 2004) provides assistance in designing the studies using 10-100 CFU of the challenge organisms.
A major concern of many QC workers is if the changes in the harmonized chapter will necessitate revalidation of existing assays to meet the requirements of the harmonized test. There are several considerations that might lead to revalidation – a required change in media, in volume of material required for testing, in general testing conditions. It is difficult to determine whether all product types would require revalidation, and so a summary table is provided (Table 2) describing the critical aspects of the current Microbial Limits Tests (Enumeration) and the draft harmonization text. The summaries provided in Table 2 are only meant as an aid, the decision as to whether or not revalidation is necessary rests with each individual facility for their particular products.

Streaking for Single Colonies: An Essential First Step in Microbial Identification

From the outset, let’s admit that the current state of microbial identification is a little confusing. We can ID by traditional biochemical tests (API Strips) or by elegant refinements of the traditional methods (for example, the Vitek 2 Compact). We can identify microorganisms by carbohydrate utilization (Biolog systems) or by the GC pattern of the cell’s fatty acids (Sherlock System). If you want to go genotypic, then you currently have a choice between the Dupont RiboPrinter or Applied Biosystems MicroSeq systems. Your identification (genus and species) may well depend on which system you use as there is no objective standard, and each system is reliant on its proprietary database to assign an identification to the data.
Virtually all of these systems require a preliminary Gram stain to accurately identify the sample. The Gram stain requires a relatively fresh culture for best results (PMF Newsletter, Feb. 2006). This is the first reason to restreak for single colonies after isolation of a contaminant. However, each system also is dependent on the presentation of a monoclonal sample for accurate results. In fact, only one of these systems provides you with enough information to recognize if you have a contaminated (polyclonal) sample (no, I am not going to tell which one it is).
The basic fact is that acceptable microbiological practice (I am not even going to say “good lab practice” or “best lab practice” but “acceptable” or, if you prefer, “adequate”) requires streaking for well isolated, single colonies of good health for identification purposes. This is not difficult.
The best starting material is a relatively “clean looking” colony on your primary plate. This colony should not show obvious signs of being multiple pinprick colonies that merged into a single CFU. Using a sterile loop, sample from the center of the colony and begin a heavy streak onto a new plate of appropriate agar media. This is quadrant #1 (see accompanying figure).
Streaking in quadrant #1 (and all subsequent streaking events) should be in the same direction, with the same part of the loop in contact with the agar. After the completion of the streaking in this quadrant, the loop should be resterilized (thoroughly flamed or discarded for a new, sterile disposable loop) and the plate streaked into quadrant #2 by drawing the fresh loop across two or three lines in quadrant #1. This should be done once or twice, then subsequent streaks performed without touching any of the previous line in the agar surface. The loop is resterilized, and the process is repeated in quadrants #3 and #4, each time the loop becoming contaminated by drawing it across a few lines in the previous quadrant. The idea is a successive dilution of the level of CFU in each quadrant, on each successive line after the initial inoculation in the prior quadrant. The plate is then incubated overnight for colony growth. Single, well-isolated colonies should be evident in quadrant #4 or quadrant #3. Care should be taken not to accidentally contaminate the colony when harvesting.
One note of caution. This streaking for single colony isolates should be conducted a second time if the original plate was heavily contaminated, or if there are multiple colony morphologies evident on this initial streaked plate. The integrity of the microbial identification process requires a monoclonal colony (a colony that is from a single bacterial strain).
Remember – the only assurance you have of a correct identification is proper preparation of the monoclonal sample. To this concern, the final isolation plate should never be used as a storage device – the single well-isolated colony chosen should be restreaked on a separate plate or agar-slant tube for storage.
This seems like a lot of work, and requires an additional day (at least) to the turn-around time for identification of a contaminant when compared to the time required if single-colony isolation is omitted from the process. However, if microbial identification is attempted directly from the colony on the primary test plate (environmental monitoring or bioburden plate) any resultant microbial identification must be suspect as there is no assurance that you are working with a pure culture. When auditing your microbiology lab (or your contract lab), check to see if an SOP is in place requiring this step, and also check the refrigerators and incubators to see if you can find evidence that this is, in fact, occurring. It is an unfortunately common practice to omit this essential step in microbial identification in an ill-advised attempt to save time and money. However, as there are few quality controls possible on the microbial identification process, you have to build the quality into the process to avoid the GIGO phenomenon.
We are in a period of high regulatory interest in environmental monitoring identifications (as part of aseptic production controls), and in the demonstration of absence of objectionable microorganisms in nonsterile finished drug products. This is not the time (if there ever was one) to save a few pennies by omitting a step necessary to the accurate and confident identification of a microbial colony.

Counting Colonies

Who Cares?
What is the fuss about in determining the number of colony forming units? After all, the CFU is only an estimate of the number of cells present. It is a skewed estimate at best as the only cells able to form colonies are those that can grow under the conditions of the test (incubation media, temperature, time, oxygen conditions, etc). Even among that group of microorganisms a colony does not represent a single cell, but rather cells that happened to be well separated on the plate and so can be distinguished after growth. A colony could arise from one cell, or several thousand. So why the fuss?
One reason for concern is that microbiology has a well-deserved reputation for being highly variable. Our lax attention to precision and accuracy in our measurements helps further this perception. We have allowed specifications for environmental monitoring, raw material bioburden, in-process bioburden and finished product bioburden to be imposed by regulation without regard for the ability of the method to support those specifications.
A second reason for concern is that now we are trying to introduce alternate microbiological methods into the lab. Being obsessive by training, we are trying to exceed measures of accuracy and precision in this exercise that the traditional methods cannot come close to matching. A good example of this is the Pharm Eur “Precision” requirement for an alternate method (quantification) to have a Relative Standard Deviation (RSD) in the range of 10-15% (1). While you might get lucky and hit this with dilutions whose plate counts are in the 150-250 CFU/plate range, - at lower plate counts this target value imposed by regulation will virtually guarantee a long, difficult and quite possibly unsuccessful, validation exercise.
Countable Range on a Plate
Literature
The general ranges in common acceptance for countable numbers of colonies on a plate are 30 – 300 and 25 – 250. The origin of those ranges is worth examination. Breed and Dotterrer published a seminal paper on this topic in 1916 (2). They set out to determine the “limit in the number of colonies that may be allowed to grow on a plate without introducing serious errors…in connection with the proposed revisions of standard methods of milk analysis.” They note that “the kind of bacteria in the material under examination will have an influence on the size of the colonies, and consequently, on the number that can develop on a plate.” They also note that food supply can be an issue, that colonies close to each other on the plate may merge, and that neighbor colonies may inhibit growth or conversely stimulate growth. “Because of these and other difficulties certain plates in any series made from a given sample are more satisfactory for use in computing a total than are others. The matter of selecting plates to be used in computing a count becomes therefore a matter requiring considerable judgment.”
Breed and Dotterrer chose their countable plates from triplicate platings of each dilution, requiring acceptable plates to be within 20% of the average. On this analysis, plates with more than 400 CFU were unsatisfactory, as were those of less than 30 CFU, with best results in the range of 50-200 CFU/plate.
The major paper from Tomasiewicz et al (3) provides an excellent review of the continued evolution of the appropriate number of CFU per plate from milk. They took data from colony counts of raw milk from three different experiments (each dilution plated in triplicate) and used to determine a mean-squared-error of the estimate for all plates. Their recommendation at the end of the study was for a countable range of 25-250 CFU/plate in triplicate. It is interesting to note that although the authors note that CFU follow a Poisson distribution, no mention is made of any data transformation used to approximate a normal distribution prior to the use of normal statistical analytical tools. Tomasiewicz et al provide excellent cautionary advice:
“The data presented herein are not necessarily applicable to other systems. For automated equipment, the optimum range may well vary with the instrument…Furthermore, even if automation is not used appropriate numbers of colonies that should be on a countable plate can very widely, depending on many other variables. With soil fungi for example…”
The compendia have recently harmonized a microbial enumeration test (4), and in this test recommend that the technician “Select the plates corresponding to a given dilution and showing the highest number of colonies less than 250 for TAMC and 50 for TYMC.” In determination of the resistance of biological indicators, USP recommends a range of “20 to 300 colonies, but not less than 6” (5). However, the most complete description of the countable range is found in the informational chapter <1227> (6):
“The accepted range for countable colonies on a standard agar plate is between 25 and 250 for most bacteria and Candida albicans. This range was established in the food industry for counting coliform bacteria in milk. The range is acceptable for compendial organisms, except for fungi. It is not optimal for counting all environmental monitoring isolates. The recommended range for Aspergillus niger is between 8 to 80 cfu per plate. The use of membrane filtration to recover challenge organisms, or the use of environmental isolates as challenge organisms in the antimicrobial effectiveness testing, requires validation of the countable range.”
ASTM provides countable ranges of 20-80 CFU/membrane, 20-200 for spread plates and 30-300 for pour plates (7). The FDA Bacterial Analytical Manual (BAM) recommends 25-250 CFU/plate as a countable range (8).
Upper Limit
The upper limit of plate counts is dependent on a number of factors, as described previously. The major issues include the colony size and behavior (swarming?), and the surface area of the plate. The size particularly comes into play with plating a membrane for determination of CFU as the surface area of that membrane is so much smaller than that of a standard plate.
TNTC can be reported out several ways. ASTM (7) recommends reporting this out as >”upper limit”. For example, a 1:10 dilution with more than 200 CFU on a spread plate would be reported as “>2,000 CFU/mL (or gram). FDA’s BAM recommends counting the colonies from the dilution with plates giving counts closest to 250, counting a portion of the plate, estimating the total number and then using that number as the Estimated Aerobic Count. It is not clear to the author how this is greatly superior to guessing. In my opinion this is an invalid plating and needs to be done correctly at a later date (note I am strenuously avoiding the use of the word retest. This result invalidates the plating and therefore the test was not performed correctly.) I know this is a hardship to the lab, who were trying to reduce the plating load initially by not plating out sufficient dilutions. However, making a mistake initially is not a reasonable excuse to avoid doing it correctly after the mistake is recognized. If the lab wishes to use this “estimated count” it should, at a minimum, have it clearly described in their “counting CFU” SOP with a rationale as to when the plate counts are not critical and can be estimated in this fashion.
There are methods available if you should want to accurately determine the upper limit for a unique plating surface or a unique colony type. One is presented in the USP informational chapter <1227> (5) which is based on a pair-wise comparison of counts from a dilution series. This is based on the assumption that at the upper limit the observed numbers of CFU will fall off the expected numbers at some point (see Figure 1). This divergence will become significant at some point – that defines the upper limit of CFU/plate.
Figure 1. Difference between Expected and Observed CFU with Increasing Numbers

Lower Limit
A central concern in this determination is the reporting of the Limit of Quantification (which is what we are really interested in reporting) against the Limit of Detection (1 CFU). This is an important distinction if we are being held to specifications in the lower range.
ASTM recommendations focus on the LOD, and urge the user to report that answer out if no colonies are recovered (ie <10 CFU/mL for a 1:10 dilution) (7). If countable colonies are present, but below the countable range, count them anyway and report an estimated count.
USP (6) does not have a specific recommendation on how to report out these low numbers, but does note “Lower counting thresholds for the greatest dilution plating in series must be justified.”
FDA BAM (8) recommends a different reporting format. In the FDA BAM method, all counts are recorded in the raw data, but the information is reported out as ). This leads to graphs such as in Figure 2 which shows us that as the CFU/plate drops below the countable range, the error as a percent of the mean increases rapidly. This confusion between the Limit of Detection and the Limit of Quantification for plate counts has led to some very difficult situations (as discussed below).
Figure 2. Increase in Error with Decreasing Numbers
Unusual Situations
What About Two Dilutions with Countable Colonies?
Ideally you would never see two separate dilutions with counts in the countable range, as the countable ranges cover a ten-fold range of CFU. However, this is microbiology. ASTM recommendations (7) urge you to take both dilutions into account, determining the CFU/mL (or gram) separately for each, then averaging the results for the final result. Breed and Dotterrer (2) also used several dilutions if the numbers fit the QC requirements (see below). FDA BAM has no recommendations in this situation.
While the argument can be made to use all counts, this is a stronger argument if triplicate plates are used and QC limits are in place to discard erroneous plates.
A strong argument can also be made to take the dilution providing the larger number of CFU in the countable range. This approach minimizes two concerns, that the errors in the estimates increase with increasing serial dilutions, and that the error in the estimate increases with decreasing plate counts. Use of the smaller dilution (eg 1:10 vs 1:100) could be justified from this perspective.
Whichever method used should be documented and justified in the “Counting CFU” SOP.
What about QC Limits on Replicate Plate Counts?
Periodically there are recommendations to establish Quality Control limits on replicate plate counts. Breed and Dotterrer in their 1916 paper (2) required valid plate counts from triplicate plates to provide estimates of CFU/mL within 20% of the mean. In other words, all plates were counted, each plate’s CFU count was used to estimate the original CFU/mL, then each estimate was evaluated. If the individual plate’s estimate was within 20% of the mean, it was deemed acceptable. This method is not practical in the QC lab.
Establishment of QC limits for plate counts works best if you have at least three replicate plates for each dilution. The average of the dilution replicates can be determined, variant counts (hopefully no more than one plate per triplicate plating) discarded and the final average determined. If you try this with duplicate plates you frequently end up with trying to average the results of one plate. While this method looks good on paper, the prudent lab manager will evaluate some historical data before instituting it as a practice.
The method used to QC individual plate counts, if used, should be documented and justified in SOP, along with the response to finding variant counts.
Can I plate 10 1 mL samples to plate a Total of One 10 mL Sample?
There have been suggestions that a larger volume of material may be plated across several plates, and the results reported out for the larger volume. For example, plating 10 1 mL samples on 10 different plates, and then reporting it as if a 10 mL sample was plated. This approach is flawed in that it ignores several sources of variability in plating including sampling error, plating errors, growth/incubation error and counting errors (9, 10). The correct interpretation for this situation that you have just plated 1 mL ten times, not 10 mL once. The numbers might be averaged, they cannot be added.
Rounding and Averaging
To discuss this we need to determine what the significant figures might be in the measure. For raw colony counts, common practice determines that the CFU observed determine the significant figure, and that the average is one decimal to the right of that number (sticklers for accuracy will report the geometric mean rather than the arithmetic mean given the Poisson distribution followed by CFU). In reporting, it is common practice to report out as scientific notation using two significant figures. This requires rounding.
USP (11) and ASTM (7) both round up at five if 5 is the number to the right of the last significant figure. FDA BAM has a more elaborate scheme, rounding up if the number is 6 or higher, down if 4 or lower. If the number is 5, BAM looks to the next number to the right and rounds up if it is odd, down if it is even.
This is one of those situations where you want every-one to do the calculations the same way (I am hard pressed to come up with a situation in a lab where you want everyone to do it differently). Be sure to include direction and its justification in the “Counting CFU” SOP if it does not already exist in a separate SOP.
Impact on Specifications and Environmental Monitoring Control Levels
We are back to the question of WHO CARES?
If you are faced with a finished product bioburden of NMT (Not More Than) 100 CFU/gram, and your method suitability study requires a 1:100 dilution of the product to overcome any antimicrobial effects, then how are you to test it? Common practice is to perform the 1:100 dilution, perform a pour plate of 1 mL in duplicate and if 2 colonies grow on each plate, the product fails specification. This common practice is scientifically unsupportable – it confused the Limit of Detection with the Limit of Quantification for the plate count method.
Let’s take a look at environmental monitoring alert and action levels for aseptically produced products. Hussong and Madsen (12) recently published a thoughtful review of this topic where they argue that the levels of acceptable CFU for many room classifications are below the noise level of plate count technology (eg in the range of 1-2 CFU/m3). In addition, environmental data can be extremely variable, much more so than controlled lab studies as the numbers of microorganisms, the physiological state of the isolates, even the species are completely out of the control of the investigator. In addition the numbers do not conform to a normal distribution as there are sporadic counts with a count of “zero” CFU predominating. They conclude that since the numbers are unreliable, the trend in the data is the only important consideration, and that EM counts cannot be used for product release criteria. A separate treatment of this subject was presented by Farrington (13) who argues that the relationship between EM data and finished product quality is a widely held, but unproven belief, compounded by the problems in accuracy with the low counts generated by plate count methodology.
Conclusions
In conclusion, all methods have limitations. One of the major limitations to the plate count method is the relatively narrow countable range (generally considered to be 25-250 CFU bacteria on a standard petri dish). The currently prevailing confusion between the Limit of Detection (1 CFU) and Limit of Quantification (25 CFU) for the plate count method creates a larger degree of variability in microbiology data than is necessary. An unfortunate regulatory trend in recent years is to establish expectations (specifications, limits, levels) for data generated by the plate count method that the accuracy of the method cannot support. This is a real opportunity for modification of current practice to approach the goal of “science-based regulations”.

How to Determine if an Organism is “Objectionable

since the microbial limits tests do not address themselves to “objectionable” microorganisms, how is the manufacturer to determine if there are “objectionables” in a lot of product awaiting release? One approach is suggested by FDA - once all organisms grown in the total count studies (total aerobic as well as total yeast and mold) are identified, a qualified microbiologist would conduct a risk analysis on the presence of that organism in that medication (4). This risk analysis should incorporate a minimum of four separate analyses: Absolute numbers of organisms seen Microorganism’s Characteristics Product Characteristics Potential Impact on Patients
Absolute Number of Organisms Seen
Although high numbers of non-pathogenic organisms may not pose a health hazard, they may affect product efficacy and/or physical /chemical stability. An unusually high number of organisms seen in the product may also indicate a problem during the manufacturing process, or an issue with a raw material. The high bacterial counts may indicate that the microorganisms are thriving in the product. If a preserved product this could indicate that the product’s preservative system is not functioning or worse, the preservative was missing or incorrectly formulated.
The Characteristics of the Microorganism
The characteristics of the microorganism can be determined by a search of textbooks, or library work, by internet searches, or a combination of all of these. It is always a good idea to remember that you are interested in the microbiology of the situation – do not restrict the search to pharmaceutical sources as most of the best information will come from food, environmental, clinical and perhaps cosmetic microbiology sources in addition to the pharmaceutical field.
During this search look for synonyms of the organisms current name. With the widespread use of genetic techniques in taxonomy the names of some organisms have undergone multiple changes. The national culture collections are a good source of synonyms and all name variants should be researched.
First of all, determine if the organism is a known pathogen. A good place to start on this search is the FDA web site the “Bad Bug Book.” This is only a guide, but a good one (5). One approach is to do a preliminary evaluation for any organism that appears on the FDA/CFSAN list and immediately classify that organism as “objectionable.” However, it is also important to consider the route of administration and the susceptible population in this evaluation.
A second characteristic of the microorganism that must be taken into account is the potential for the organism to cause spoilage of the product. Make a list of substances used by the microorganism for growth. This can be from the literature, or from the identification equipment. For example, the Vitek 2 Compact will provide an extensive list of compounds the microorganism can metabolize, the Biolog a list of carbohydrates utilized, etc. Use the information gained during the identification of the organism. Compare these to the product formulation for potential issues. A microorganism is also objectionable if it has the potential to degrade the product on stability. Evaluate the microorganism’s tolerance to unusual conditions: low or high pH high salt concentration high sugar concentration (osmotic conditions) Low water activity Growth temperature, etc. It can also be useful to determine if the microorganism has a recognized proclivity for harboring plasmid-mediated antibiotic resistance. This is a special concern in regards to horizontal transmission of the trait within an vulnerable patient population.
Product Characteristics
The dosage form is important to consider. Is the product anhydrous or water based? This can have an effect on the ability of microorganisms to proliferate. Does it have sufficient free water to support microbial growth (6, 7). Is the container designed to minimize contamination and subsequent spoilage? Closure design can have a major effect on in-use stability of a product. Is the container adequately designed to retard access to the environment, and to prevent contamination from the environment. Give special consideration to the likelihood of an anhydrous medication’s exposure to water, providing the potential for microbial proliferation. The route of administration is also important. A medication orally administered can tolerate some microorganisms that would be disastrous in a medication meant to be applied topically to abraded skin or to rashes. Similarly, some microorganisms that could be tolerated in a topical would cause severe distress to a patient if taken orally. Inhalants, although not required to be sterile, are a particularly sensitive area and great care should be taken in classifying any contaminate as “non-objectionable.” Other product-related considerations should include a review of the production records and the environmental monitoring trends, A review of field complaints is also useful (is this contaminant one that causes eventual returns?).
Patient Population
Finally, a consideration of the targeted patient population is in order. The manufacturer cannot control, and should be held accountable, for patient abuse of a product or off-label use of the product by physicians. However, reasonable use of the product should be considered and part of the risk analysis. Are patient populations that are likely to use this product at increased risk if exposed to the particular microorganism?
Summary
The FDA’s concern with non-sterile dosage format is that the product not contain “objectionable” organisms. This FDA concern has been made clear since the 1970’s. However, many companies continue to mistakenly believe that if their non-sterile product meets the requirements in USP, it will be safe from FDA dispute. This not the case. The manufacturer is responsible for all contents of his drug product. Should question arise over the appropriateness of a particular organism, the manufacturer is expected to have a justification for the presence of that organism, preferably as part of the batch release document. Presented here is a brief description of some factors to consider in determining if an organism is objectionable. These considerations include: Absolute number of organisms present Microorganism characteristics Product characteristics Patient Population These are not meant to be a comprehensive listing of all issues, but rather a starting point for the non-sterile manufacturer to use in establishing their program to qualify finished product bioburden

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