Tuesday, October 4, 2011

Conventional Methods for Diagnosis of Infectious Diseases

Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world’s biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood.
Causes of infectious diseases:
The microbial causes of human diseases are classified into theses groups:
Infection may be endogenous or exogenous.
Endogenous infections: the microorganism (usually a bacterium) is a component of the patient's endogenous flora. Endogenous infections can occur when the microorganism is aspirated from the upper to the lower respiratory tract or when it penetrates the skin or mucosal barrier as a result of trauma or surgery.
Exogenous infections: the microorganism is acquired from the environment (e.g., from soil or water) or from another person or an animal.
The ability to control such microbial infections is largely dependent on the ability to detect these aetiological agents in the clinical microbiology laboratory (Millar et al., 2003)
Diagnoses of Infectious diseases
Diagnosis of infectious diseases in clinical microbiological laboratory through
- Conventional methods
Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture) or indirectly (eg, identifying antibodies to the organism
-molecular methods.

Specimen Selection, Collection and Processing
A- Specimen quantity
Specimens selected for microbiologic examination should reflect the disease process and be collected in sufficient quantity to allow complete microbiologic examination. The number of microorganisms per milliliter of a body fluid or per gram of tissue is highly variable, ranging from less than 1 to 108 or 1010 colony-forming units (CFU).
Swabs, although popular for specimen collection, frequently yield too small a specimen for accurate microbiologic examination and should be used only to collect material from the skin and mucous membrane
Specimen collection is important. The wrong type of swab can produce false-negative results. Wooden-shafted swabs are toxic to some viruses. Cotton-tipped swabs are toxic for some bacteria and chlamydiae. Blood cultures require decontamination and disinfection of the skin (eg, povidone iodine swab, allowed to dry, removed with 70% alcohol). Multiple samples, each from a different site are generally used; they are taken nearly simultaneously with fever spikes if possible. Normal flora of skin and mucous membranes that grow in only a single blood sample are usually interpreted as contamination.
If a blood specimen is obtained from a central line, a peripheral blood specimen should also be obtained to help differentiate systemic bacteremia from catheter infection. Cultures from infected catheters generally turn positive more quickly and contain more organisms than simultaneously drawn peripheral blood cultures. Some fungi, particularly molds (eg, Aspergillus sp), usually cannot be cultured from blood.
The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations.
B-Prevention of contamination of a specimen
Because skin and mucous membranes have a large and diverse endogenou flora, every effort must be made to minimize specimen contamination during collection. Contamination may be avoided by various means:
The skin can be disinfected before aspirating or incising a lesion.
The contaminated area may be bypassed altogether. Examples of such approaches are trans tracheal puncture with aspiration of lower respiratory secretions or supra pubic bladder puncture with aspiration of urine.
It is often impossible to collect an uncontaminated specimen, and decontamination procedures, cultures on selective media, or quantitative cultures must be used.
Specimens collected by invasive techniques, particularly those obtained intra operatively, require special attention. Enough tissue must be obtained for both histopathologic and microbiologic examination.
C-Time of collection
If possible, specimens should be collected before the administration of antibiotics. Above all, close communication between the clinician and the microbiologist is essential to ensure that appropriate specimens are selected and collected and that they are appropriate
The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations.
1- Laboratory methods for diagnosis of infectious diseases
A-Direct method:
They are Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture)
1- Microscopy
Microscopy can be done quickly, but accuracy depends on the experience of the microscopist and quality of equipment. Regulations often limit physicians' use of microscopy for diagnostic purposes outside a certified laboratory (Siqueira, J. Fet al., 2005)
Most specimens are treated with stains that color pathogens, causing them to stand out from the background, although wet mounts of unstained samples can be used to detect fungi, parasites (including helminth eggs and larvae), vaginal clue cells, motile organisms (eg, Trichomonas), and syphilis (via darkfield microscopy). Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms (Fredricks and Relman, 1999).
The clinician orders a stain based on the likely pathogens, but no stain is 100% specific. Most samples are treated with Gram stain and, if mycobacteria are suspected, an acid-fast stain. However, some pathogens are not easily visible using these stains; if these pathogens are suspected, different stains or other identification methods are required. Because microscopic detection usually requires a microbe concentration of about 1 × 105/mL, most body fluid specimens (eg, CSF) are concentrated (eg, by centrifugation) before examination.

Types of stains which commonly used.
Gram stain: The Gram stain classifies bacteria according to whether they retain crystal violet stain (gram-positive—blue) or not (gram-negative—red) and highlights cell morphology (eg, bacilli, cocci) and cell arrangement (eg, clumps, chains, diploids). Such characteristics can direct antibiotic therapy pending definitive identification. To do a Gram stain, technicians heat-fix specimen material to a slide and stain it by sequential exposure to Gram's crystal violet, iodine, decolorizer, and counterstain (typically safranin).
Acid-fast and moderate (modified) acid-fast stains: These stains are used to identify acid-fast organisms (Mycobacterium sp) and moderately acid-fast organisms (primarily Nocardia sp). These stains are also useful for staining Rhodococcus and related genera, as well as oocysts of some parasites (eg, Cryptosporidium).
Although detection of mycobacteria in sputum requires only about 5, 000 to 10, 000 organisms/mL, mycobacteria are often present in lower levels, so sensitivity is limited. Usually, several mL of sputum are decontaminated with Na hydroxide and concentrated by centrifugation for acid-fast staining. Specificity is better, although some moderately acid-fast organisms are difficult to distinguish from mycobacteria.
Fluorescent stains: These stains allow detection at lower concentrations (1 × 104 cells/mL). Examples are acridine orange (bacteria and fungi), auramine-rhodamine and auramine O (mycobacteria), and calcofluor white (fungi, especially dermatophytes).
Coupling a fluorescent dye to an antibody directed at a pathogen (direct or indirect immunofluorescence) should theoretically increase sensitivity and specificity. However, these tests are difficult to read and interpret, and few (eg, Pneumocystis and Legionella direct fluorescent antibody tests) are commercially available and commonly used.
India ink (colloidal carbon) stain: This stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, CSF sediment). The background field, rather than the organism itself, is stained, which makes any capsule around the organism visible as a halo. In CSF, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated.
Wright's stain and Giemsa stain: These stains are used for detection of parasites in blood, Histoplasma capsulatum in phagocytes and tissue cells, intracellular inclusions formed by viruses and chlamydia, trophozoites of Pneumocystis jiroveci, and some intracellular bacteria.
Trichrome stain (Gomori-Wheatley stain) and iron hematoxylin stain:
These stains are used to detect intestinal protozoa.
The Gomori-Wheatley stain is used to detect microsporidia. It may miss helminth eggs and larvae and does not reliably identify Cryptosporidium. Fungi and human cells take up the stain.
The iron hematoxylin stain differentially stains cells, cell inclusions, and nuclei. Helminth eggs may stain too dark to permit identification.

Disadvantages of Microscopic methods :
Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species.
Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator.
Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible.
Limited specificity is because our inability to speciate micro-organisms based on their morphology and staining patterns.

2-Culture methods
Culture is microbial growth on or in a nutritional solid or liquid medium; increased numbers of organisms simplify identification. Culture also facilitates testing of antimicrobial susceptibility (Relman DA., 2002)
Communication with the laboratory is essential. Although most specimens are placed on general purpose media (e g, blood or chocolate agar), some pathogens require inclusion of specific nutrients and inhibitors or other special conditions (Wade W., 2002)
For more than a century, cultivation using artificial growth media has been the standard diagnostic test in infectious diseases. The microbiota associated with different sites in the human body has been extensively and frequently defined by studies using cultivation approaches.
The success in cultivation of important pathogenic bacteria probably led microbiologists to feel satisfied with and optimistic about their results and to recognize that there is no death of known pathogens (Relman, 1992 and Wade W, 2002).
But should we be so complacent with what we know about human pathogens? Making micro-organisms grow under laboratory conditions presupposes some knowledge of their growth requirements. Nevertheless, very little is known about the specific growth factors that are utilized by innumerous micro-organisms to survive in virtually all habitats, including within the human body (Wade, 2002).
A huge proportion of the microbial species in nature are difficult to be tamed in the laboratory. Certain bacteria are fastidious or even impossible to cultivate. Some well-known human pathogens, such as Mycobacterium leprae and Treponema pallidum continue to defy scientists regarding their cultivation under laboratory conditions (Fredricks and Relman, 1999)
Culture Anaerobic bacteria
Should not be cultured from sites where they are normal flora because differentiation of pathogens from normal flora may be impossible. Specimens must be shielded from air, which can be difficult. For swab specimens, anaerobic transport media are available. Specimens collected with a syringe (eg, abscess contents) should be transported in the syringe.
Culture of Mycobacteria
Mycobacteria are difficult to culture. Specimens containing normal flora (eg, sputum) must first be decontaminated and concentrated. Mycobacterium tuberculosis and some other mycobacteria grow slowly. Growth of M. tuberculosis is typically faster in liquid than in solid media; routine use of automated systems with liquid media can result in growth within 2 wk vs ≥ 4 wk on solid media such as Lowenstein-Jensen agar. In addition, few organisms may be present in a specimen. Multiple specimens from the same site may help maximize yield. Specimens should be allowed to grow for 8 wk before being discarded. If an atypical mycobacterium is suspected, the laboratory should be notified (Kevin C. Hazen, PhD 2009)
Culture of Viruses
Viruses are generally cultured from swabs and tissue specimens usually transported in media that contain antibacterial and antifungal agents. Specimens are inoculated onto tissue cultures that support the suspected virus and inhibit all other microbes.
Viruses that are highly labile (eg, varicella zoster) should be inoculated onto tissue cultures within 1 h of collection. Standard tissue cultures are most sensitive. Rapid tissue cultures (shell vials) may provide more rapid results. Some common viruses cannot be detected using routine culture methods and require alternative methods for diagnosis (eg, enzyme immunoassay for Epstein-Barr virus, hepatitis B and E viruses, HIV, and human T-lymphotrophic virus; serologic tests for hepatitis A and D viruses; nucleic acid–based methods for HIV) (Kevin C. Hazen, PhD 2009)

Culture of fungi
Fungi specimens obtained from non sterile sites must be inoculated onto media containing antibacterial agents. Specimens should be allowed to grow for 4 wk before being discarded.
Advantages of cultivation approaches:
The main advantages of cultivation approaches are related to their broad-range nature, which makes it possible to identify a great variety of microbial species in a sample, including those that are not being sought after. Still, cultivation makes it possible to determine antimicrobial susceptibilities of the isolates and to study their physiology and pathogenicity.
Disadvantages of cultivation approaches:
Cultivation-based identification approaches have several limitations:
They are costly.
They can take several days to weeks to identify some fastidious anaerobic bacteria (that can delay antimicrobial treatment).
They have a very low sensitivity (particularly for fastidious anaerobic bacteria).
Their specificity may be also low and is dependent on the experience of the microbiologist.
They have strict dependence on the mode of sample transport.
Finally, the impossibility of cultivating a large number of bacterial species as well as the difficulties in identifying many cultivable species represent the major drawbacks of cultivation-based approaches.
There are many possible reasons for bacterial unculturability, (Kell & Young, 2000 and Wade, 2002).
Obviously, if micro-organisms can not be cultivated, they can not be identified by phenotype-based methods. While we stay relatively ignorant on the requirements of many bacteria to grow, identification methods that are not based on bacterial culturability are required. (Wade, 2004)
It is worth pointing out the fact that a given species is uncultivable does not necessarily imply that the same species will remain indefinitely impossible to cultivate. For instance, a myriad of strict anaerobic bacteria were uncultivable 100 years ago, but further developments in cultivation techniques have helped solve this problem. There is a growing trend to develop approaches and culture media that allow cultivation of previously uncultivated bacteria. Strategies may rely on application of cultivation procedures that better mimic conditions existing in the natural habitat from which the samples were obtained.
Recent efforts to accomplish this have met with some success by including the following: the use of agar media with little or no added nutrients; relatively lengthy periods of incubation (more than 30 days); and inclusion of substances that are typical of the natural environment in the artificial growth media (Kell &Young, 2002 and Stevenson et al., 2004).
However, it is not unreasonable to surmise that a huge number of species will remain uncultivable for years to come.

B- Indirect methods
They are testes that identify antibodies to the organism
-Immunological Methods
Immunological methods are based on the specificity of antigen-antibody reaction. It can detect micro-organisms directly or indirectly, the latter by detecting host immunoglobulins specific to the target micro- organism. The enzyme-linked immunosorbent assay (ELISA) and the direct or indirect immunofluorescence tests are the most commonly used immunological methods for microbial identification (Siqueira, J. Fet al., 2005)
Immunologic tests use an antigen to detect antibodies to a pathogen or use an antibody to detect an antigen of the pathogen in the patient's specimen. Handling varies, but if testing is to be delayed, the specimen should typically be refrigerated or frozen to prevent overgrowth of bacterial contaminants.
Latex particle agglutination, co agglutination, and enzyme-linked immunosorbent assay (ELISA) are the most frequently used techniques in the clinical laboratory (Baron et al., 1995).
Agglutination tests:
In agglutination tests (eg, latex agglutination, coaggregation), a particle (latex bead or bacterium) is coupled to a reagent antigen or antibody. The resulting particle complex is mixed with the specimen (eg, CSF, serum); if the target antibody or antigen is present in the specimen, it cross-links the particles, producing measurable agglutination.
If results are positive, the body fluid is serially diluted and tested. Agglutination with more dilute solutions indicates higher concentrations of the target antigen or antibody. The titer is correctly reported as the reciprocal of the most dilute solution yielding agglutination; eg, 32 indicates that agglutination occurred in a solution diluted to 1/32 of the starting concentration. see figure (18)
Usually, agglutination tests are rapid but less sensitive than many other methods. They can also determine serotypes of some bacteria.

Figure (18) Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.
Complement fixation:
This test measures complement-consuming (complement-fixing) antibody in serum or CSF. The test is used for diagnosis of some viral and fungal infections, particularly coccidioidomycosis. The specimen is incubated with known quantities of complement and the antigen that is the target of the antibody being measured. The degree of complement fixation indicates the relative quantity of the antibody in the specimen. The test can measure IgM and IgG antibody titers or can be modified to detect certain antigens. It is accurate but has limited applications, is labor intensive, and requires numerous controls.
Enzyme immunoassays:
These tests use antibodies linked to enzymes to detect antigens and to detect and quantify antibodies. The enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are examples. Because sensitivities of most enzyme immunoassays are high, they are usually used for screening. Titers can be determined by serially diluting the specimen as for agglutination tests.
Test sensitivities, although usually high, can vary, sometimes according to patient age, microbial serotype, specimen type, or stage of clinical disease.
Precipitation tests:
These tests measure an antigen or antibody in body fluids by the degree of visible precipitation of antigen-antibody complexes within a gel (agarose) or in solution. There are many types of precipitation tests (eg, Ouchterlony double diffusion, counter immunoelectrophoresis), but their applications are limited. Usually, a blood specimen is mixed with test antigen to detect patient antibodies, most often in suspected fungal infection or pyogenic meningitis. Because a positive result requires a large amount of antibody or antigen, sensitivity is low.
Western blot test:
This test detects antimicrobial antibodies in the patient's sample (eg, serum, other body fluid) by their reaction with target antigens (eg, viral components) that have been immobilized onto a membrane by blotting.
The Western blot typically has good sensitivity, although often less than that of screening tests such as ELISA, but generally is highly specific. Thus, it is usually used to confirm a positive result obtained with a screening test.
Technical modifications of the Western blot are the line immunoassay (LIA); the recombinant immunoblot assay (RIBA), which use synthetic or recombinant-produced antigens; and immunochromatographic assays, which can rapidly screen specimens for specific microbial antigens or patient antibodies.
Advantages of immunological methods :
They take no more than a few hours to identify a microbial species.
They can detect dead micro-organisms.
They can be easily standardized.
They have low cost (Sixou, 2003).
Disadvantages of immunological methods:
They can detect only target species.
They have low sensitivity (about 104 cells).
Their specificity is variable and depends on types of antibodies used.
They can detect dead micro-organisms (Zambon & Haraszthy, 2000 and Sixou, 2003).
- Serodiagnosis
Infection may be diagnosed by an antibody response to the infecting microorganism. This approach is especially useful when the suspected microbial agent either cannot be isolated in culture by any known method or can be isolated in culture only with great difficulty.
The diagnosis of hepatitis virus and Epstein-Barr virus infections can be made only serologically, since neither can be isolated in any known cell culture system. Although human immunodeficiency virus type 1 (HIV-1) can be isolated in cell cultures, the technique is demanding and requires special containment facilities. HIV-1 infection is usually diagnosed by detection of antibodies to the virus (Murray et al., 1995).
Disadvantages of Serodiagnosis :
There is usually a lag between the onset of infection and the development of antibodies to the infecting microorganism.
Although IgM antibodies may appear relatively rapidly, it is usually necessary to obtain acute- and convalescent-phase serum samples to look for a rising titer of IgG antibodies to the suspected pathogen. In some instances the presence of a high antibody titer when the patient is initially seen is diagnostic.
However, the high titer may reflect a past infection, and the current infection may have an entirely different cause.
The immunosuppressed patients may be unable to mount an antibody response.

Antimicrobial Susceptibility Tests
Susceptibility tests determine a microbe's vulnerability to antimicrobial drugs by exposing a standardized concentration of organism to specific concentrations of antimicrobial drugs (Jorgensen, J. and M.J. Ferraro. 1998)
The term susceptible means that the microorganism is inhibited by a concentration of antimicrobial agent that can be attained in blood with the normally recommended dose of the antimicrobial agent and implies that an infection caused by this microorganism may be appropriately treated with the antimicrobial agent. The term resistant indicates that the microorganism is resistant to concentrations of the antimicrobial agent that can be attained with normal doses and implies that an infection caused by this microorganism could not be successfully treated with this antimicrobial agent (Woods & Washington, 1995).
Susceptibility testing can be done for bacteria, fungi, and viruses. For some organisms, results obtained with one drug predict results with similar drugs. Thus, not all potentially useful drugs are tested.
Aim of Antimicrobial Susceptibility tests
The purpose of performing antimicrobial susceptibility testing (AST) is to provide in vitro data to help ensure that appropriate and adequate antimicrobial therapy is used to optimize treatment outcomes. In addition, the AST data generated daily can be statistically analyzed on an annual basis to generate an antibiogram that reflects the antimicrobial susceptibility and resistance patterns of important pathogens that prevail in a particular hospital. These hospital-specific AST epidemiologic data provide valuable guidance to the clinicians for the appropriate selection of empiric therapy, prior to the availability of culture and susceptibility results that often takes 2 to 3 days (Walsh, T.R et al., 2000)
Indications for routine susceptibility tests
A susceptibility test may be performed in the clinical laboratory for two main purposes:
To guide the clinician in selecting the best antimicrobial agent for an individual patient;
To accumulate epidemiological information on the resistance of microorganisms of public health importance within the community.
Susceptibility tests as a guide for treatment
Susceptibility tests should never be performed on contaminants or commensals belonging to the normal flora, or on other organisms that have no causal relationship to the infectious process. For example, the presence of Escherichia coli in the urine in less than significant numbers is not to be regarded as causing infection, and it would be useless and even misleading to perform an antibiogram.
Routine susceptibility tests are not indicated in the following situations:
When the causative organism belongs to a species with predictable susceptibility to specific drugs. This is the case for Streptococcus pyogenes and Neisseria meningitidis, which are still generally susceptible to penicillin (However, there have recently been a few reports of sporadic occurrences of penicillin-resistant meningococci.) It is also the case for faecal streptococci (enterococci), which, with few exceptions, are susceptible to ampicillin. If resistance of these microorganisms is suspected on clinical grounds, representative strains should be submitted to a competent reference laboratory.
If the causative organism is slow-growing or fastidious and requires enriched media, e.g., Haemophilus influenzae and Neisseria gonorrhoeae, disc-diffusion susceptibility tests may give unreliable results.
In uncomplicated intestinal infections caused by salmonellae (other than S. typhi or S. paratyphi), susceptibility tests are not routinely needed. Antibiotic treatment of such infections is not justified, even with drugs showing in vitro activity. There is now ample evidence that antimicrobial treatment of common salmonella gastroenteritis (and indeed of most types of diarrhoeal disease of unknown etiology) is of no clinical benefit to the patient. Paradoxically, antibiotics prolong the excretion and dissemination of salmonellae and may lead to the selection of resistant variants.
Methods of anti microbial susceptibility tests:
A-Phenotypic methods.
General principles of antimicrobial susceptibility testing
Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial agent to inhibit bacterial growth in vitro. This ability may be estimated by either the dilution method or the diffusion method
Microbial susceptibility testing guidelines are based on MIC determinations. These are correlated with the pharmacological properties of the antimicrobial drug and the clinical results when the drug is used. In MIC determinations, a bacteriological breakpoint is usually drawn between susceptible and resistant organisms. This breakpoint has been defined as the minimum concentration at which the drug is predicted to be clinically effective against the etiologic agents.

A- Disk Diffusion method (Kirby-Bauer)
This is the standard AST used in most bacteriology laboratories in the country.
In the disk diffusion susceptibility test; a standardized inoculum of the organism is swabbed onto the surface of a Muller-Hinton agar plate, then filter paper disks impregnated with antimicrobial agents are placed on the agar. After overnight incubation at 35°C, the diameter of the zone of inhibition (ZI) of bacterial growth around each disk is measured. (Jorgensen, J. H., and J. D. Turnidge.,2007)
The diffusion of the antimicrobial agent into the seeded culture media results in a gradient of the antimicrobial. When the concentration of the antimicrobial becomes so diluted that it can no longer inhibit the growth of the test bacterium, the zone of inhibition is demarcated. The diameter of this zone of inhibition around the antimicrobial disk is related to minimum inhibitory concentration (MIC) for that particular bacterium/antimicrobial combination; the zone of inhibition correlates inversely with the MIC of the test bacterium.
Generally, the larger the zone of inhibition, the lower the concentration of antimicrobial required to inhibit the growth of the organisms. However, this depends on the concentration of antibiotic in the disk and its diffusibility.
Based on the zones of inhibition a qualitative report of "susceptible, " “intermediate" or "resistant" can be determined for rapidly growing non-fastidious aerobic bacteria
Disk diffusion tests based solely on the presence or absence of a zone of inhibition without regard to the size of the zone of inhibition are not acceptable AST methodology (CLSI., 2006)
Disk diffusion is straightforward to perform, reproducible, and does not require expensive equipment. Its main advantages are:
i) low cost,
ii) easy in modifying test antimicrobial disks when required,
iii) can be used as a screening test against large numbers of isolates,
iv) can identify a subset of isolates for further testing by other methods, such as determination of MICs.
Disadvantages &Limitations
Some organisms susceptibility testing by disc diffusion method is not applicable as
Organisms that are fastidious, slow growing or have special requirement for growth e.g anaerobes.
Mycobacterial &fungal infection need special techniques.
Certain antibiotics cannot be accurately tested e.g polymyxins
Manual measurement of zones of inhibition may be time-consuming. Automated zone-reading devices are available that can be integrated with laboratory reporting and data-handling systems. (CLSI.2006)
B-Dilution method:
The aim of the broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial that inhibits the growth of the bacterium being tested (MIC, usually expressed in mg/ml or mg/litre).
However, the MIC does not always represent an absolute value. The ‘true’ MIC is a point between the lowest test concentration that inhibits the growth of the bacterium and the next lower test concentration.
Therefore, MIC determinations performed using a dilution series may be considered to have an inherent variation of one dilution.
Antimicrobial ranges should encompass both the interpretive criteria (susceptible, intermediate and resistant) for a specific bacterium/antibiotic combination and appropriate quality control reference organisms.
Antimicrobial susceptibility dilution methods appear to be more reproducible and quantitative than agar disk diffusion. However, antibiotics are usually tested in doubling dilutions, which can produce inexact MIC data.
(Jorgensen, J. H., and J. D. Turnidge.,2007)
Broth dilution
In the broth dilution MIC method, various concentrations of an antimicrobial drug are inoculated with a standard suspension of test bacteria. Following an overnight incubation at 35°C, the MIC is determined by observing the lowest concentration of the drug that will inhibit visible growth of the test bacteria. For full range MIC testing, 5- 8 concentrations representing a therapeutically achievable range for an antimicrobial are usually tested. Recent modifications, however, provide only 1-3 concentrations of each drug and results are reported.
The broth dilution method can be performed
- macrodilution
The broth dilution method can be performed in tubes containing a minimum volume of 2 ml
- microdilution
It performed in smaller volumes using microtitration plates. Numerous microtitre plates containing prediluted antibiotics within the wells are commercially available.
The use of identical lots in microdilution plates may assist in the minimisation of variation that may arise due to the preparation and dilution of the antimicrobials from different laboratories.
Due to the fact that most broth microdilution antimicrobial test panels are prepared commercially, this method is less flexible than agar dilution or disk diffusion in adjusting to the changing needs of the surveillance/monitoring programme.
MIC test method has been well standardized for most fastidious rapidly growing bacteria.
More tests can be done at the same time by limiting dilution steps.
The broth microdilution MIC test method has been well standardized for most fastidious rapidly growing bacteria.
Because the purchase of antimicrobial plates and associated equipment may be costly, this methodology may not be feasible for some laboratories (CLSI. 2007)

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