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Diagnostic Microbiology
Laboratory evidence to support the diagnosis of an infectious disease may be based on one or more of the following: direct examination of specimens using microscopic or antigen detection techniques, isolation of microorganisms in culture, serologic testing, host gene expression patterns, or molecular detection of an organism, resistance determinant, or virulence factor. Some additional roles of the clinical microbiology laboratory include performing antimicrobial susceptibility testing and supporting hospital infection prevention in the detection and characterization of pathogens associated with nosocomial infections.
Specimen Collection
The success of a diagnostic microbiology assay, that is, detection of a pathogen if present, is directly linked to specimen collection techniques. In general, this means collecting the correct specimen type for the disease or condition in question and promptly transporting the specimen to the laboratory for analysis. Although swab specimens may be necessary for some conditions, in general a swab is a suboptimal specimen. A swab is only able to hold a very small amount of specimen (approximately 100 µL), and, using a traditional swab, only a small fraction of organisms that are absorbed onto a swab will be released back into the culture. Flocked swabs coupled with transport medium improve organism recovery. However, when possible, fluid or tissue should be submitted to the laboratory for analysis. If anaerobic infection is suspected, the sample should be transported in appropriate medium to preserve viability of anaerobic bacteria. For the recovery of some organism types, such as viruses and Neisseria gonorrhoeae , specific transport media may be required. Considerations specific to the collection of blood cultures are addressed in the blood culture section.
Laboratory Diagnosis of Bacterial and Fungal Infections
Although the scope and availability of molecular methods for detection of bacterial and fungal pathogens have increased rapidly, the diagnosis of many of these infections depends on microscopic detection of organisms or cultivation of organisms on culture media .
Microscopy
The Gram stain is an extremely valuable diagnostic technique to provide rapid and inexpensive information regarding the absence or presence of inflammatory cells and organisms in clinical specimens. For some specimen types, the presence of inflammatory and epithelial cells is used to judge the suitability of a specimen for culture. For example, the presence of >10 epithelial cells per low-power field in a sputum specimen is highly suggestive of a specimen contaminated with oral secretions. In addition, a preliminary assessment of the etiologic agent can be made based on the morphology (e.g., cocci vs rods) and stain reaction (e.g., gram-positive isolates are purple; gram-negative isolates are red) of the microorganisms. However, a negative Gram stain does not rule out infection, since 10 4 to 10 5 microorganisms per milliliter (mL) in the specimen are required for detection by this method.
In addition to the Gram stain, many other stains are used in microbiology, both to detect organisms and to help infer their identity ( Table 195.1 ).
Table 195.1
Stains Used for Microscopic Examination
| TYPE OF STAIN | CLINICAL USE |
|---|---|
| Gram stain | Stains bacteria (with differentiation of gram-positive and gram-negative organisms), fungi, leukocytes, and epithelial cells. |
| Potassium hydroxide (KOH) | A 10% solution dissolves cellular and organic debris and facilitates detection of fungal elements in clinical specimens. |
| Calcofluor white stain | Nonspecific fluorochrome that binds to cellulose and chitin in fungal cell walls, can be combined with 10% KOH to dissolve cellular material. |
| Ziehl-Neelsen and Kinyoun stains | Acid-fast stains, using basic carbolfuchsin, followed by acid-alcohol decolorization and methylene blue counterstaining. |
| Acid-fast organisms (e.g., Mycobacterium ) resist decolorization and stain pink. | |
| A weaker decolorizing agent is used for partially acid-fast organisms (e.g., Nocardia, Cryptosporidium, Cyclospora, Isospora ). | |
| Auramine-rhodamine stain | Acid-fast stain using fluorochromes that bind to mycolic acid in mycobacterial cell walls and resist acid-alcohol decolorization; usually performed directly on clinical specimens. |
| Acid-fast organisms stain orange-yellow against a black background. | |
| Acridine orange stain | Fluorescent dye that intercalates into DNA, used to aid in differentiation of organisms from debris during direct specimen examination, and also for detection of organisms that are not visible with Gram stain. |
| Bacteria and fungi stain orange, and background cellular material stains green. | |
| Lugol iodine stain | Added to wet preparations of fecal specimens for ova and parasites to enhance contrast of the internal structures (nuclei, glycogen vacuoles). |
| Wright and Giemsa stains | Primarily for detecting blood parasites ( Plasmodium, Babesia, and Leishmania ), detection of amoeba in preparations of cerebrospinal fluid, and fungi in tissues (yeasts, Histoplasma ) |
| Trichrome stain | Stains stool specimens for identification of protozoa. |
| Direct fluorescent-antibody stain | Used for direct detection of a variety of organisms in clinical specimens by using specific fluorescein-labeled antibodies (e.g., Pneumocystis jiroveci, many viruses). |
Isolation and Identification
The approach to isolation of microorganisms in a clinical specimen will vary depending on the body site and pathogen suspected. For body sites that are usually sterile, such as cerebrospinal fluid, nutrient-rich media such as sheep blood agar and chocolate agar are used to aid in the recovery of fastidious pathogens. In contrast, stool specimens contain abundant amounts of commensal bacteria, and thus to isolate pathogens, selective and differential media must be used. Selective media will inhibit the growth of some organisms to aid in isolation of suspect pathogens; differential media rely on growth characteristics or carbohydrate assimilation characteristics to impart a growth pattern that differentiates organisms. MacConkey agar supports growth of gram-negative rods while suppressing gram-positive organisms, and a color change in the media from clear to pink distinguishes lactose-fermenting organisms from other gram-negative rods. Special media, such as Sabouraud dextrose agar and inhibitory mold agar, are used to recover fungi in clinical specimens. Many pathogens, including Bartonella , Bordetella pertussis , Legionella , Mycoplasma , some Vibrio spp., and certain fungal pathogens such as Malassezia furfur , require specialized growth media or incubation conditions. Consultation with the laboratory is advised when these pathogens are suspected.
Once an organism is recovered in culture, additional testing is performed to identify the isolate. Confirmation of microbial identity has classically been performed using tests that rely on the phenotypic properties of an isolate; examples include coagulase activity, carbohydrate assimilation patterns, indole production, and motility. However, phenotypic methods are not able to resolve all organisms to species level, and they require incubation time. In some instances, sequence-based identification may be necessary. For bacteria, this is usually based on sequence analysis of the bacterial 16S rRNA gene. This gene is a molecular chronometer that is highly conserved within a species but variable between species; as such, it is an excellent resource for organism identification.
Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry ( MALDI-TOF MS ) is a rapid and accurate technique that is based on generating a protein fingerprint of an organism and comparing that fingerprint to a library of known organisms to produce an identification. This method can identify bacteria or yeast that have been recovered in culture within minutes, and the consumable costs for these analyses are minimal. However, this methodology currently lacks the ability to resolve polymicrobial samples, and the biomass required for successful MALDI-TOF MS analysis generally precludes analysis directly from clinical specimens.
Blood Culture
The detection of microbes in blood culture specimens of patients with bloodstream infection is one of the most important functions of the clinical microbiology laboratory. Most blood cultures are performed by collecting blood into bottles of nutrient-rich broth to facilitate the growth of bacteria or yeast. Blood cultures are frequently submitted as a set that includes an aerobic and an anaerobic bottle, although in children, especially neonates, typically only an aerobic bottle is used. Some blood culture media contain resins or other agents to help neutralize antibiotics that may be present in the patients’ blood. Blood culture bottles are then placed into an automated blood culture incubator that will monitor the blood culture bottle at regular intervals for evidence of growth. Once the instrument detects evidence of microbial growth, an alarm alerts the laboratory. Approximately 80% of blood cultures that will ultimately be positive are identified within the 1st 24 hr of incubation. A portion of broth from a blood culture bottle that has signaled positive is then gram-stained and subsequently inoculated onto appropriate growth media so that the organism can be isolated and identified. Numerous preanalytical variables can influence the accuracy of blood culture results. To facilitate accurate interpretation of a positive blood culture, a minimum of 2 blood cultures drawn from different sites should be collected whenever possible. Growth of an organism that is part of the normal skin flora from a single blood culture raises concern that the isolate resulted from contamination of the culture.
To maximize detection of bloodstream infection, up to 4 blood cultures should be collected over 24 -hr. Proper skin antisepsis is essential before blood collection. Chlorhexidine is frequently used for this purpose, but alcohol is also used. If blood is collected through an indwelling line, proper antisepsis before collection is also important. The practice of obtaining blood for culture from intravascular catheters without accompanying peripheral venous blood cultures should be discouraged, because it is difficult to determine the significance of coagulase-negative staphylococci and other skin flora or environmental organisms isolated from blood obtained from line cultures. Differential time to positivity of 2 hr or more between paired blood cultures drawn simultaneously from a catheter and peripheral vein has been cited as an indicator of catheter-related bloodstream infection.
The volume of blood collected is also an important factor in the recovery of bloodstream pathogens, especially as the number of organisms per milliliter of blood in sepsis may be low (<10 colony-forming units/mL). The optimal amount of blood to collect from a pediatric patient varies depending on the weight of the child. The Clinical and Laboratory Standards Institute (CLSI) and Cumitech provide guidance on the amount of blood that is safe to collect from children of different sizes. For children from 3 to <12 kg, 3-5 mL is suggested; from 12 to <36 kg, 5-10 mL; from 36-50 kg, 10-15 mL, and >50 kg, 20 mL.
A number of rapid diagnostic assays can be performed directly on positive blood culture broth to identify pathogens frequently associated with bacteremia and some antimicrobial resistance determinants. Most of these rapid diagnostic assays are based on nucleic acid detection techniques. For example, the Verigene system can identify staphylococcal, streptococcal, and enterococcal species, as well as mecA and vanA genes, in positive blood culture broth in approximately 2 hr using the gram-positive blood culture panel. After specimen preparation to concentrate microorganisms and remove residual broth and blood from the blood culture specimen, MALDI-TOF MS can also be performed on blood culture broth that is positive for growth of microorganisms. These assays can help shorten the interval between a positive blood culture and definitive organism identification, with the goal of early optimization of antimicrobial therapy.
Detection of mycobacteria and some filamentous fungi (e.g., Histoplasma capsulatum ) from the bloodstream is maximized using lysis-centrifugation techniques, such as the Isolator system (Wampole, Cranbury, NJ).
Cerebrospinal Fluid Culture
Cerebrospinal fluid (CSF) should be transported quickly to the laboratory and then cytocentrifuged to concentrate organisms for microscopic examination. CSF is routinely cultured on blood agar and chocolate agar, which support the growth of common pathogens causing meningitis. If tuberculosis is suspected, cultures for mycobacteria should be specifically requested. Culture of larger volumes of CSF (>10 mL) significantly improves yield of mycobacteria.
Historically, rapid antigen detection tests for bacterial pathogens such as Haemophilus influenzae type b and Streptococcus pneumoniae were used to attempt to detect organisms in CSF without the need for culture. These techniques lack sensitivity and in some cases specificity. A cytospin Gram stain is as sensitive as bacterial antigen tests for detection of microorganisms in CSF. In contrast, the cryptococcal antigen test can be useful when cryptococcal meningitis is suspected. Historically, India ink preparations were used to detect Cryptococcus in CSF, but this method is insensitive compared with the antigen detection assay.
In the postvaccine era, the epidemiology of infectious meningitis is rapidly changing, and acute bacterial meningitis is now a relatively infrequent event in North America. Many CSF infections are associated with shunts or other hardware, and Propionibacterium and coagulase-negative staphylococci are the organisms most frequently isolated from shunt infections. The laboratory should include media to facilitate the growth of Propionibacterium in CSF specimens received from neurosurgery patients.
Urine Culture
Urine for culture (including colony count) can be obtained by collecting clean-voided midstream specimens, by catheterization, or by suprapubic aspiration. Urine samples collected by placing bags on the perineum are unacceptable for culture because samples are often contaminated. Rapid transport of unpreserved urine to the laboratory (<2 hr) is imperative, and delay in transport or plating of specimens renders colony counts unreliable. Refrigeration or urine transport devices with boric acid preservative may be used when delay is unavoidable.
The specific colony counts used to define growth in a urine culture as significant are somewhat controversial and vary by laboratory. Urine obtained by suprapubic aspirate is normally sterile, and thus any organism growth is typically considered significant. Urine collected by catheterization is likely to reflect infection if there are ≥10 3 to 10 4 organisms/mL. In general, clean-voided urine is considered abnormal if ≥10 4 to 10 5 organisms/mL are present, although culture interpretation can be variable depending on the patient's age and the clinical setting.
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