How is infection diagnosed?
Gram Stain and Culture
In order to perform a gram stain and culture, obtain a sample of exudate or body fluid. The diagnosis can be made in in minutes (gram stain) to hours to days (culture) for bacteria, weeks for mycobacteria
requires collection of the specimen with the appropriate devices requires filling out the proper laboratory request forms (the lab is not able to "do everything" on every submitted sample) requires setting up the cultures on the appropriate media
Colony Counts
For urine cultures, it is useful to know the relative numbers of bacteria as well as their identification, since most methods of urine collection yield a ssample with some contamination. In the example below, a calibrated loop with 0.001 mL of the urine sample is plated onto culture media and incubated. The number of colonies present is multiplied by 1000. In this example, there are over 100,000 colonies/mL.
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More rapid diagnosis of urinary tract infection can be done with methods to detect bacterial growth in specimens by photometry or bioluminescence. In general, such methods work well when large numbers of organisms are present. Urine microscopic examination can be performed on a fresh uncentrifuged urine sample with gram stain. This method is very specific, but the absence of visible bacteria does not exclude infection, particularly when fewer organisms are present. Pyuria with WBCs can be identified by microscopy, and is more sensitive than the dipstick leukocyte esterase in fresh urine samples. A so-called "sterile pyuria" may occur with infection from agents not detected by routine microscopy or culture: Chlamydia trachomatis, Ureaplasma urealyticum, and Mycobacterium tuberculosis.
Antimicrobial Sensitivity Testing
Antimicrobial sensitivity testing of the organism can be performed. The Kirby-Bauer plate shown below illustrates the diffusion disk method in which antibiotic-impregnated disks are placed on a culture plate with the organism, and the zone of inhibition of growth is measured, compared to controls. In general, less than 2 mm of inhibition defines resistance, while a large zone of inhibition suggests sensitivity. Smaller zones imply intermediate sensitivity. In the example below, the organism is sensitive to Amikacin and Penicillin, with intermediate sensitivity to Gentamicin and Cefotaxime, with resistance to Methicillin, Vancomycin, and Nafcillin.
The series of test tubes shown below demonstrate the minimum inhibitory concentration (MIC) method. 1 ml of broth containing the organism is added to each tube; the tubes contain serial dilutions of an antibiotic, ranging from 0.5 microgram/mL to 64 microgram/mL. A control tube contains no antibiotic. The MIC is the tube with the lowest level (highest dilution) of antibiotic in which no growth occurs.
In the example below, the MIC is 2 microgram/mL for ampicillin.
The MIC is an in vitro assay. One may want to know what effectiveness an antibiotic has in the patient's serum. A minimum inhibitory dilution (MID) may be performed by taking a patient serum sample, making serial dilutions, and placing those patient serum dilutions into broth growing the organism isolated from the patient.
In the example below, the MID is performed to correlate with doses of the antibiotic given and measured in the patient's serum as "peak" and "trough" levels just after and just before administration of the dose, respectively. The MID is at a 1:4 dilution for the trough level of amikacin, and at 1:32 for the peak level. In general, an MID of 1:32 suggests good antibiotic effectiveness.
Trough
Peak
The usefulness of antibiotic combinations showing synergy of bactericidal activity can be demostrated similarly. In the example below, the wells inoculated with an organism contain increasing concentrations of two antimicrobial agents. While a concentration of 32 micrograms/mL is required to inhibit growth by gentamicin alone, a combination of 4 micrograms/mL of gentamicin with 1 microgram/mL of penicillin will inhibit growth.
Serology
Serology is used for organisms that are difficult (viruses) to culture;
answer in hours to days can be "paired sera" with an initial "acute" titer followed by a "convalescent" titer in about 2 weeks to determine if there is a rise in antibody to a specific organism can differentiate the titer according to either IgG or IgM antibody. IgG
antibodies can persist for months to years, so they only give an indication of
some past infection. IgM antibodies tend to appear only with acute infection.
Thus, hepatitis A virus serologies are generally ordered as IgG and IgM to
determine if there has been recent infection. View serologic techniques
Microscopy
Molecular Probes
Molecular probes are markers for the genetic material in micro-organisms
Antimicrobial resistance in bacteria may occur via horizontal spread of resistance genes, such as those expressing drug-insensitive variants of the target enzymes dihydropteroate synthase and dihydrofolate reductase, for sulfonamide and trimethoprim, respectively. Two genes, sul1 and sul2, mediated by transposons and plasmids, and expressing dihydropteroate synthases highly resistant to sulfonamide, have been found. For trimethoprim, almost twenty phylogenetically different resistance genes, expressing druginsensitive dihydrofolate reductases have been characterized. They are efficiently spread as cassettes in integrons, and on transposons and plasmids.
In the example below, isolates of E. coli have undergone DNA extraction following by restriction endonuclease digestion and gel electrophoresis to determine plasmid profiles. The profile of the organism in lane 3 is clearly different, and this may help explain patterns of antibiotic resistance. Though plasmid profile typing is not routinely performed, it can be an epidemiologic tool.
Epidemiology in infectious diseases
Statistical review of infectious disease cases
prospectively: each state has a list of diseases that MUST be reported to
the health department by YOU the health care worker. Statistics can be compiled
in regard to incidence and distribution (age, geography, season, etc) retrospectively: medical records can be reviewed, laboratory reports
analyzed, patients followed up, or anatomic pathology material (surgical
pathology, autopsy records) reviewed
Patterns of infectious disease can be determined over time
some infections (such as rotavirus) are more common in the spring and
summer, while others (such as influenza in adults or respiratory syncytial virus in infants and children) are more common in winter; this alerts you to the need to be aware of such seasonal changes and what the more likely diagnoses will be in your patients, as shown in the example below.
In the next example below, note the increase in reported cases for an infectious disease for one month, suggesting some specific event occurred, such as a food borne outbreak.
geographic localization of infections help to determine where prevention
strategies or treatment programs should be employed, or help to localize a
source of infection (such as a contaminated well or a restaurant) a sharp increase in cases can alert the health department to the need to
identify a potential epidemic in the making
Epidemiologists and public health department workers can collect additional data:
interview infected persons to determine how, when, and where they became
infected follow up contacts of infected persons (such as sexual partners of persons with sexually transmitted diseases (STD's) such as gonorrhea, syphilis, and HIV test food and water, or test animal vectors, for presence of infectious agents
A course of action is taken (quarantine, immunization program, closure of a business or facility, specific treatment of contacts or a subset of the population, etc.)
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