Diagnostic medical microbiology is concerned with the etiologic diagnosis of infection. Laboratory procedures used in the diagnosis of infectious disease in humans include the following:

1. 
   

   Morphologic identification of the agent in stains of specimens or sections of tissues (light and electron microscopy).

   
   
2. 
   

   Detection of the agent in patient specimens by antigen testing (latex agglutination, enzyme immunoassay, etc) or nucleic acid testing (nucleic acid hybridization, polymerase chain reaction [PCR], sequencing, etc).

   
   
3. 
   

   Culture isolation and identification of the agent. Susceptibility testing of the agent by culture or nucleic acid methods, where appropriate.

   
   
4. 
   

   Demonstration of meaningful antibody or cell-mediated immune responses to an infectious agent.

In the field of infectious diseases, laboratory test results depend largely on the quality of the specimen, the timing and the care with which it is collected and transported, and the technical proficiency and experience of laboratory personnel. Although physicians should be competent to perform a few simple, crucial microbiologic tests (perform direct wet mounts of certain specimens, make a Gram-stained smear and examine it microscopically, and streak a culture plate), the technical details of the more involved procedures are usually left to trained microbiologists. Physicians who deal with infectious processes must know when and how to take specimens, what laboratory examinations to request, and how to interpret the results.

This chapter discusses diagnostic microbiology for bacterial, fungal, and viral diseases. The diagnosis of parasitic infections is discussed in Chapter 46.

Diagnostic microbiology encompasses the detection and characterization of thousands of agents that cause or are associated with infectious diseases. The techniques used to characterize infectious agents vary greatly depending on the clinical syndrome and the type of agent being considered, be it virus, bacterium, fungus, or parasite. Because no single test will permit isolation or characterization of all potential pathogens, clinical information is much more important for diagnostic microbiology than it is for clinical chemistry or hematology. The clinician must make a tentative diagnosis rather than wait until laboratory results are available. When tests are requested, the physician should inform the laboratory staff of the tentative diagnosis (type of infection or infectious agent suspected). Proper labeling of specimens includes such clinical data as well as the patient’s identifying data (at least two methods of definitive identification) and the requesting physician’s name and pertinent contact information.

Many pathogenic microorganisms grow slowly, and days or even weeks may elapse before they are isolated and identified. Treatment cannot be deferred until this process is complete. After obtaining the proper specimens and informing the laboratory of the tentative clinical diagnosis, the clinician should begin treatment with drugs aimed at the organism thought to be responsible for the patient’s illness. As the laboratory staff begins to obtain results, they will inform health care providers, who can then reevaluate the diagnosis and clinical course of the patient and perhaps make changes in the therapeutic program. This “feedback” information from the laboratory consists of preliminary reports of the results of individual steps in the isolation and identification of the causative agent.

Specimens

Laboratory examination usually includes microscopic study of fresh unstained and stained materials and preparation of cultures with conditions suitable for growth of a wide variety of microorganisms, including the type of organism most likely to be causative based on clinical evidence. If a microorganism is isolated, complete identification may then be pursued. Isolated microorganisms may be tested for susceptibility to antimicrobial drugs. When significant pathogens are isolated before treatment, follow-up laboratory examinations during and after treatment may be appropriate.

A properly collected specimen is the single most important step in the diagnosis of an infection, because the results of diagnostic tests for infectious diseases depend on the selection, timing, and method of collection of specimens. Bacteria and fungi grow and die, are susceptible to many chemicals, and can be found at different anatomic sites and in different body fluids and tissues during the course of infectious diseases. Because isolation of the agent is so important in the formulation of a diagnosis, the specimen must be obtained from the site most likely to yield the agent at that particular stage of illness and must be handled in such a way as to favor the agent’s survival and growth. For each type of specimen, suggestions for optimal handling are given in the following paragraphs and in the later section on diagnosis by anatomic site.

Recovery of bacteria and fungi is most significant if the agent is isolated from a site normally devoid of microorganisms (a normally sterile area). Any type of microorganism cultured from blood, cerebrospinal fluid (CSF), joint fluid, the pleural cavity, or peritoneal cavity is a significant diagnostic finding. Conversely, many parts of the body have normal microbiota (Chapter 10) that may be altered by endogenous or exogenous influences. Recovery of potential pathogens from the respiratory, gastrointestinal, or genitourinary tracts; from wounds; or from the skin must be considered in the context of the normal microbiota of each particular site. Microbiologic data must be correlated with clinical information in order to arrive at a meaningful interpretation of the results.

A few general rules apply to all specimens:

1. 
   

   The quantity of material must be adequate.

   
   
2. 
   

   The sample should be representative of the infectious process (eg, sputum, not saliva; pus from the underlying lesion, not from its sinus tract; a swab from the depth of the wound, not from its surface).

   
   
3. 
   

   Contamination of the specimen must be avoided by using only sterile equipment and aseptic technique.

   
   
4. 
   

   The specimen must be taken to the laboratory and examined promptly. Special transport media may be needed.

   
   
5. 
   

   Meaningful specimens to diagnose bacterial and fungal infections must be secured before antimicrobial drugs are administered. If antimicrobial drugs are given before specimens are taken for microbiologic study, drug therapy may have to be stopped and repeat specimens obtained several days later.

The type of specimen to be examined is determined by the presenting clinical picture. If symptoms or signs point to involvement of one organ system, specimens are obtained from that source. In the absence of localizing signs or symptoms, repeated blood samples for culturing are taken first, and specimens from other sites are then considered in sequence, depending in part on the likelihood of involvement of a given organ system in a given patient and in part on the ease of obtaining specimens.

Microscopy and Stains

Microscopic examination of stained or unstained specimens is a relatively simple and inexpensive, but much less sensitive method than culture for detection of small numbers of bacteria. A specimen must contain at least 10⁵ organisms per milliliter before it is likely that organisms will be seen on a smear. Liquid medium containing 10⁵ organisms per milliliter does not appear turbid to the eye. Specimens containing 10²–10³ organisms per milliliter produce growth on solid media, and those containing 10 or fewer bacteria per milliliter may produce growth in liquid media.

Gram staining is a very useful procedure in diagnostic microbiology. Most specimens submitted when bacterial infection is suspected should be smeared on glass slides, Gram-stained, and examined microscopically. The materials and method for Gram staining are outlined in Table 47-1. On microscopic examination, the Gram reaction (purple-blue indicates gram-positive organisms; red, gram-negative) and morphology (shape: cocci, rods, fusiform, or other; see Chapter 2) of bacteria should be noted. In addition, the presence or absence of inflammatory cells and the type of cell are important to note and quantify. Likewise, the presence of material that does not appear inflammatory, such as squamous epithelial cells obtained from a respiratory sample or wound, may be useful for determining the adequacy of the sample collection. The appearance of bacteria on Gram-stained smears does not permit identification of species. Reports of gram-positive cocci in chains are suggestive of, but not definitive for, streptococcal species; gram-positive cocci in clusters suggest a staphylococcal species. Gram-negative rods can be large, small, or even coccobacillary. Some nonviable gram-positive bacteria can stain as gram negative. Typically, bacterial morphology has been defined using organisms grown on agar. However, bacteria in body fluids or tissue can have highly variable morphology.

Specimens submitted for examination for mycobacteria should be stained for acid-fast organisms. The most sensitive fluorescent stains for mycobacteria detection, such as auramine-rhodamine, should be used. Confirmation of a positive fluorescent stain is usually performed using one of the nonfluorescent acid-fast stains, either Ziehl-Neelsen stain or Kinyoun stain (Table 47-1). These stains can be used as alternatives to the fluorescent stains for mycobacteria in laboratories that lack fluorescence microscopy (see Chapter 23). Immunofluorescent antibody (IF) staining is useful in the identification of many microorganisms. Such procedures are more specific than other staining techniques but also more cumbersome to perform. The fluorescein-labeled antibodies in common use are made from antisera produced by injecting animals with whole organisms or complex antigen mixtures. The resultant polyclonal antibodies may react with multiple antigens on the organism that was injected and may also cross-react with antigens of other microorganisms or possibly with human cells in the specimen. Quality control is important to minimize nonspecific IF staining. Use of monoclonal antibodies may circumvent the problem of nonspecific staining. IF staining is most useful in confirming the presence of specific organisms such as Bordetella pertussis or Legionella pneumophila in colonies isolated on culture media. The use of direct IF staining on specimens from patients is more difficult and less specific and is largely being replaced by nucleic acid amplification techniques (NAATs).

Stains such as calcofluor white, methenamine silver, Giemsa, and occasionally periodic acid-Schiff (PAS) and others are used for tissues and other specimens in which fungi or parasites are present. Such stains are not specific for given microorganisms, but they may define structures so that morphologic criteria can be used for identification. Calcofluor white binds to cellulose and chitin in the cell walls of fungi and fluoresces under long-wavelength ultraviolet light. It may demonstrate morphology that is diagnostic of the species (eg, spherules with endospores in Coccidioides immitis infection). Pneumocystis jirovecii cysts are identified morphologically in silver-stained specimens. PAS is used to stain tissue sections when fungal infection is suspected. After primary isolation of fungi, stains such as lactophenol cotton blue are used to distinguish fungal growth and to identify organisms by their morphology.

Specimens to be examined for fungi can be examined unstained after treatment with a solution of 10% potassium hydroxide, which breaks down the tissue surrounding the fungal mycelia to allow a better view of the hyphal forms. Phase contrast microscopy is sometimes useful in unstained specimens. Dark-field microscopy is used to detect Treponema pallidum in material from primary or secondary syphilitic lesions or other spirochetes such as Leptospira.

Culture Systems

For diagnostic bacteriology, it is necessary to use several types of media for routine culture, particularly when the possible organisms include aerobic, facultatively anaerobic, and obligately anaerobic bacteria. The specimens and culture media used to diagnose the more common bacterial infections are listed in Table 47-2. The standard medium for specimens is blood agar, usually made with 5% sheep blood. Most aerobic and facultatively anaerobic organisms will grow on blood agar. Chocolate agar, a medium containing heated blood with or without supplements, is a second necessary medium; some organisms that do not grow on blood agar, including pathogenic Neisseria and Haemophilus, will grow on chocolate agar. A selective medium for enteric gram-negative rods (either MacConkey agar or eosin–methylene blue [EMB] agar) is a third type of medium used routinely. These agars contain indicators that allow differentiation of lactose-fermenting organisms from non–lactose-fermenting organisms. Specimens to be cultured for obligate anaerobes must be plated on anaerobic media, such as brucella agar, a highly supplemented medium with hemin and vitamin K or a selective medium containing substances that inhibit the growth of enteric gram-negative rods and facultatively anaerobic or anaerobic gram-positive cocci.

Many other specialized media are used in diagnostic bacteriology; choices depend on the clinical diagnosis and the organism under consideration. The laboratory staff selects the specific media on the basis of the information in the culture request. Thus, freshly made Bordet-Gengou or charcoal-containing medium is used to culture for B pertussis in the diagnosis of whooping cough, and other special media are used to culture for Vibrio cholerae, Corynebacterium diphtheriae, Neisseria gonorrhoeae, and Campylobacter species. For culture of mycobacteria, specialized solid and liquid media are commonly used. These media may contain inhibitors of other bacteria. Because many mycobacteria grow slowly, the cultures must be incubated and examined periodically for weeks (see Chapter 23).

Broth cultures in highly enriched media are important for back-up cultures of biopsy tissues and body fluids such as CSF. Broth cultures may give positive results when there is no growth on solid media because of the small number of bacteria present in the inoculum (see above).

Many yeasts will grow well on blood agar. Biphasic and mycelial phase fungi grow better on media designed specifically for fungi. Brain–heart infusion agar, with and without antibiotics, and inhibitory mold agar have largely replaced the traditional use of Sabouraud’s dextrose agar to grow fungi. Media made with plant and vegetable materials, the natural habitats for many fungi, also grow many fungi that cause infections. Cultures for fungi are commonly done in paired sets, one set incubated at 25–30°C and the other at 35–37°C. Table 47-3 outlines specimens and other tests to be used for the diagnosis of fungal infections.

In addition to the above standard and selective media, agars that incorporate antibiotics and chromogenic enzyme substrates that impart color to specific organisms of interest, such as methicillin-resistant Staphylococcus aureus and various Candida species, among many others, are available. These media, while more expensive, do enhance sensitivity by inhibiting background microbiota and allowing the pathogen of interest to be more easily recognized. Typically, these chromogenic agars are used for specimens such as surveillance cultures and cultures of urine.

Antigen Detection

Immunologic systems designed to detect antigens of microorganisms can be used in the diagnosis of specific infections. Immunofluorescent tests (direct and indirect fluorescent antibody tests) are one form of antigen detection and are discussed in separate sections in this chapter and in the chapters on the specific microorganisms.

Enzyme immunoassays (EIAs), including enzyme-linked immunosorbent assays (ELISA), and agglutination tests are used to detect antigens of infectious agents present in clinical specimens. The principles of these tests are reviewed briefly here.

There are many variations of EIAs to detect antigens. One commonly used format is to bind a capture antibody, specific for the antigen in question, to the wells of plastic microdilution trays. The specimen containing the antigen is incubated in the wells followed by washing of the wells. A second antibody for the antigen, labeled with enzyme, is used to detect the antigen. Addition of the substrate for the enzyme allows detection of the bound antigen by colorimetric reaction. A significant modification of EIAs is the development of immunochromatographic membrane formats for antigen detection. In this format, a nitrocellulose membrane is used to absorb the antigen from a specimen. A colored reaction appears directly on the membrane with sequential addition of conjugate followed by substrate. In some formats, the antigen is captured by bound antibody directed against the antigen. These assays have the advantage of being rapid and also frequently include a built-in internal control. EIAs are used to detect viral, bacterial, chlamydial, protozoan, and fungal antigens in a variety of specimen types such as stool, CSF, urine, and respiratory samples. Examples of these are discussed in the chapters on the specific etiologic agents.

In latex agglutination tests, an antigen-specific antibody (either polyclonal or monoclonal) is fixed to latex beads. When the clinical specimen is added to a suspension of the latex beads, the antibodies bind to the antigens on the microorganism forming a lattice structure, and agglutination of the beads occurs. Coagglutination is similar to latex agglutination except that staphylococci rich in protein A (Cowan I strain) are used instead of latex particles; coagglutination is less useful for antigen detection compared with latex agglutination but is helpful when applied to identification of bacteria in cultures such as Streptococcus pneumoniae, Neisseria meningitidis, N gonorrhoeae, and β-hemolytic streptococci.

Latex agglutination tests are primarily directed at the detection of carbohydrate antigens of encapsulated microorganisms. Antigen detection is used most often in the diagnosis of group A streptococcal pharyngitis. Detection of cryptococcal antigen is useful in the diagnosis of cryptococcal meningitis in patients with AIDS or other immunosuppressive diseases.

The sensitivity of latex agglutination tests in the diagnosis of bacterial meningitis is not better than that of Gram stain, which is approximately 100,000 bacteria per milliliter. For that reason, the latex agglutination test is not recommended for direct CSF specimen testing.

Serological Testing

Detection of specific antibodies to infectious agents can be useful for diagnosis of acute or chronic infections, and for investigating the epidemiology of infectious disease. During the course of illness, IgM antibody is first produced, followed by appearance of IgG antibody. Caution must be used when interpreting positive IgM results, as these assays demonstrate cross-reactivity and can be falsely positive. Serology is most useful when acute and convalescent sera are tested to show increases in antibody titers over time.

There are a variety of serological assays available, including direct immunofluorescence, agglutination, complement fixation (CF), EIA, and ELISA formats. There are also nonspecific immunoassays available, such as the heterophile test for EBV mononucleosis and rapid plasma reagin for syphilis. Several of these tests can measure antibody titer by performing dilutions of patient serum to determine the lowest titer at which reactivity is seen.

Western Blot Immunoassays

These assays are usually performed to detect antibodies against specific antigens of a particular organism. This method is based on the electrophoretic separation of major proteins of the organism in question in a two-dimensional agarose gel. Organisms are mechanically or chemically disrupted, and resultant solubilized antigen of the organism is placed in a polyacrylamide gel. An electric current is applied, and major proteins are separated out on the basis of size (smaller proteins travel faster). The protein bands are transferred to strips of nitrocellulose paper. Following incubation of the strips with a patient’s specimen containing antibody (usually serum), the antibodies bind to the proteins on the strip and are detected enzymatically in a fashion similar to the EIA methods described earlier. Western blot tests are used as specific tests for antibodies in HIV infection and Lyme disease.

Molecular Diagnostics

A. Nucleic Acid Hybridization Probes

The principle behind hybridization probe molecular assays is the hybridization of a characterized nucleic acid probe to a specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. For example, single-stranded probe DNA (or RNA) is used to detect complementary RNA or denatured DNA in a test specimen. The nucleic acid probe typically is labeled with enzymes, antigenic substrates, chemiluminescent molecules, or radioisotopes to facilitate detection of the hybridization product. By carefully selecting the probe or making a specific oligonucleotide and performing the hybridization under conditions of high stringency, detection of the nucleic acid in the test specimen can be extremely specific. Such assays are currently used primarily for rapid confirmation of a pathogen once growth is detected (eg, the identification of Mycobacterium tuberculosis in culture using a DNA probe). In situ hybridization involves the use of labeled DNA probes or labeled RNA probes to detect complementary nucleic acids in formalin-fixed paraffin-embedded tissues, frozen tissues, or cytologic preparations mounted on slides. Technically, this can be difficult and is usually performed in histology laboratories and not clinical microbiology laboratories. However, this technique has increased the knowledge of the biology of many infectious diseases, especially the hepatitides and oncogenic viruses, and is still useful in infectious diseases diagnosis. A novel technique that is somewhat of a modification of in situ hybridization makes use of peptide nucleic acid probes. Peptide nucleic acid probes are synthesized pieces of DNA in which the sugar phosphate backbone of DNA (normally negatively charged) is replaced by a polyamide of repetitive units (neutral charge). Individual nucleotide bases can be attached to the now neutral backbone, which allows for faster and more specific hybridization to complementary nucleic acids. Because the probes are synthetic, they are not subject to degradation by nucleases and other enzymes. These probes can be used for detection of S aureus, enterococci, certain Candida spp., and some gram-negative bacilli from positive blood culture bottles. The probe hybridization is detected by fluorescence and is called peptide nucleic acid–fluorescence in situ hybridization (PNA-FISH).

B. Bacterial Identification Using 16S rRNA Probe Hybridization

The 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in each organism. Labeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double-stranded hybrid is measured. This technique is widely used for the rapid identification of many organisms. Examples include the most common and important Mycobacterium species, C immitis, Histoplasma capsulatum, and others.

Molecular diagnostic assays that use amplification of nucleic acid have become widely used and are evolving rapidly. They have been used on a variety of sample types including direct patient specimens, positive cultures, and isolated organisms. These amplification systems fall into several basic categories as outlined below.

C. Target Amplification Systems

In these assays, the target DNA or RNA is amplified many times. The polymerase chain reaction (PCR) is used to amplify extremely small amounts of specific DNA present in a clinical specimen, making it possible to detect what were initially minute amounts of the DNA. PCR uses a thermostable DNA polymerase to produce a twofold amplification of target DNA with each temperature cycle. Conventional PCR, also referred to as end detection PCR, utilizes three sequential reactions—denaturation, annealing, and primer extension—as follows. The DNA extracted from the clinical specimen along with sequence-specific oligonucleotide primers, nucleotides, thermostable DNA polymerase, and buffer are heated to 90–95°C to denature (separate) the two strands of the target DNA. The temperature in the reaction is lowered, usually to 45–60°C depending on the primers, to allow annealing of the primers to the target DNA. Each primer is then extended by the thermostable DNA polymerase by adding nucleotides complementary to the target DNA yielding the twofold amplification. The cycle is then repeated 30–40 times to yield amplification of the target DNA segment by more than 10¹⁰-fold. The amplified segment often can be seen in an electrophoretic gel or detected by Southern blot analysis using labeled DNA probes specific for the segment or by a variety of proprietary commercial techniques. More recently, real-time PCR protocols have replaced these end detection methods (see below).

PCR can also be performed on RNA targets, which is called reverse transcriptase PCR. The enzyme reverse transcriptase is used to transcribe the RNA into complementary DNA for subsequent PCR amplification.

PCR assays are available commercially for identification of a broad range of bacterial and viral pathogens such as Chlamydia trachomatis, N gonorrhoeae, M tuberculosis, cytomegalovirus (CMV), HIV-1, hepatitis C virus, and many others. There are many other laboratory-developed PCR assays that have been implemented by individual laboratories to diagnose infections. Such assays are the tests of choice to diagnose many infections—especially when traditional culture and antigen detection techniques do not work well. Examples include testing of CSF for herpes simplex virus (HSV) to diagnose herpes encephalitis and testing of nasopharyngeal samples to diagnose B pertussis infection (whooping cough).

A major consideration for laboratories that perform PCR assays is to prevent contamination of reagents or specimens with target DNA from the environment, which can obscure the distinction between truly positive results and falsely positive ones because of the contamination.

E. Signal Amplification Techniques

These assays strengthen the signal by amplifying the label (eg, fluorochromes, enzymes) that is attached to the target nucleic acid. The branched DNA (bDNA) system has a series of primary probes and a branched secondary probe labeled with enzyme. Multiple oligonucleotide probes specific for the target RNA (or DNA) are fixed to a solid surface such as a microdilution tray. These are the capture probes. The prepared specimen is added, and the RNA molecules are attached to the capture probes on the microdilution tray. Additional target probes bind to the target but not to the tray. The enzyme-labeled bDNA amplifier probes are added and attach to the target probes. A chemiluminescent substrate is added, and light emitted is measured to quantitate the amount of target RNA present. Examples of the use of this type of assay include the quantitative measurement of HIV-1, hepatitis C virus, and hepatitis B virus.

F. Amplification Methods: Non–PCR-Based

The transcription-mediated amplification (TMA) and the nucleic acid sequence–based amplification (NASBA) systems amplify large quantities of RNA in isothermal assays that coordinately use the enzymes reverse transcriptase, RNase H, and RNA polymerase. An oligonucleotide primer containing the RNA polymerase promoter is allowed to bind to the RNA target. The reverse transcriptase makes a single-stranded cDNA copy of the RNA. The RNase H destroys the RNA of the RNA–cDNA hybrid, and a second primer anneals to the segment of cDNA. The DNA-dependent DNA polymerase activity of reverse transcriptase extends the DNA from the second primer, producing a double-stranded DNA copy, with intact RNA polymerase. The RNA polymerase then produces many copies of the single-stranded RNA. Detection of C trachomatis, N gonorrhoeae, and M tuberculosis and quantitation of HIV-1 viral loads are examples of the use of these types of assays.

Strand displacement assays (SDA) are isothermal amplification assays that employ use of restriction endonuclease and DNA polymerase. The restriction endonuclease “nicks” the DNA at specific sites allowing DNA polymerase to initiate replication at the nicks on the target molecule and simultaneously displacing the nicked strand. Displaced single strands then serve as templates for additional amplification.

Loop-mediated isothermal amplification (LAMP

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