Chapter 47: Principles of Diagnostic Medical Microbiology

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) gets its name from the fact that the final amplification product consists of a structure that contains multiple loops (repeats) of the target sequence. The reaction is isothermal and consists of autocycling strand displacement DNA synthesis using Bst DNA polymerase and four to six primers. Amplification products can be detected in real time by precipitating DNA by adding magnesium pyrophosphate to the reaction creating turbidity that can be read visually or by using a spectrophotometer. This method is very sensitive, detecting as few as 10 target copies per reaction. A commercial assay using LAMP technology for the detection of Clostridium difficile in stool samples and other pathogens in a variety of specimen types is available.

G. Real-Time PCR

Technologic advances, which have lead to “real-time amplification,” have streamlined nucleic acid amplification platforms, improved the sensitivity of amplification tests, and drastically reduced the potential for contamination. Dramatic improvements in the chemistry of nucleic acid amplification reactions have resulted in homogeneous reaction mixtures in which fluorogenic compounds are present in the same reaction tube in which the amplification occurs. A variety of fluorogenic molecules are used. These include nonspecific dyes such as SYBR green, which binds to the minor groove of double-stranded DNA, and amplicon-specific detection methods using fluorescently labeled oligonucleotide probes, which fall into three categories: TaqMan or hydrolysis probes; fluorescence energy transfer (FRET) probes; and molecular beacons. The signal from these probes is proportional to the amount of product DNA present in the reaction and is plotted against the PCR cycle. Use of a threshold value allows determination of positive and negative reactions. The signal is measured through the closed reaction tube using fluorescent detectors; hence, the assay is performed in “real time.” Since the reaction tube does not need to be opened to analyze the PCR products on a gel, there is much less risk of amplicon carry-over to the next reaction. When used with a standard curve, real-time PCR assays can be quantitative, allowing for determination of organism concentration. These assays are commonly used for viral load quantification of HIV, hepatitis C virus, hepatitis B virus, and CMV. The reader is referred to the Persing et al reference for more detailed information about real-time PCR and other molecular methods.

H. PCR-Sequencing

The product of a PCR reaction can be sequenced and compared to a database for identification of organisms or resistance mutations. PCR primers are designed to hybridize to conserved genomic regions, with the sequence of interest amplified between the primers. A variety of sequencing methods can be used, the discussion of which is beyond the scope needed here.

For bacterial identification, sequencing of the 16S rRNA gene is commonly used. This gene has highly conserved regions interspersed with variable sequences, making it ideal for amplifying and differentiating many bacterial species. Other conserved gene targets are also used for bacterial identification, including rpoB, sodA, and hsp65. Similarly, fungal identification can be performed using PCR-sequencing of 28S rRNA gene and ribosomal RNA gene internal transcribed spacer elements.

PCR-sequencing is also used for strain typing and detection of specific resistance mutations in viruses (see Diagnosis of Viral Infections section below). Its use is expanding to gene characterization in other organisms, such as the detection of certain mutations causing rifampin or isoniazid resistance in M tuberculosis.

I. Microarrays

Nucleic acid microarrays involve the use of multiple oligonucleotide probes to detect the complementary target sequence in amplified DNA or RNA. The arrays can have from tens to hundreds of thousands of probes (high-density microarrays), and yield substantial information about the genetic makeup of specific organisms. Patient samples or clinical isolates are subject to DNA amplification labeling followed by hybridization, washing, and detection of labeled DNA bound to specific probes. Microarrays can be used to detect microorganisms directly from patient samples or positive blood cultures through the use of conserved targets such as 16S ribosomal DNA probes. They can also provide genetic profiling of isolated organisms, yielding information about the genotype, virulence factors, or resistance markers present in the organism.

J. High-Throughput Sequencing

High-throughput sequencing (also known as next-generation or “deep” sequencing) involves the simultaneous sequencing of a large number of DNA molecules (known as a library). The source of the library can be an organism isolate or direct patient sample. Several different instrument platforms are available, and can generate thousands to millions of sequence reads per sample. Bioinformatic algorithms are then used to classify, assemble, and compare the sequence to known organism databases. High-throughput sequencing can be used to assemble entire organism genomes, define the microbiome, detect infectious agents, or look for low-level sequence variants, known as quasispecies. Database comparison can be used to classify the organism subtype or determine the presence of markers of drug resistance or virulence.

Mass Spectrometry

Mass spectrometry (MS), a technology used to analyze proteins or DNA, has revolutionized the approach to microbial identification in clinical laboratories. MS employs methods such as ionization radiation to disrupt material, forming charged particles that are identified in various ways on the basis of mass or mass-to-charge ratio. Applications in microbiology have been made possible by advances in technology, such as matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Several different methods are briefly described below.

MassTag PCR incorporates a tag of known mass (a library of 64 mass tags is commercially available) into the PCR product. Most frequently used in multiplex PCR reactions, the tags are released by ultraviolet (UV) irradiation and analyzed by MS. The identity of the desired target or targets is determined by the size of the tag(s).

PCR electrospray ionization mass spectrometry (PCR-ESI-MS) uses a unique principle. Briefly, for particular microbes, a set of PCR primers is designed that amplifies key regions of the microbial genome. Multiple PCR reactions are conducted in a microtiter plate for analysis of each sample, and some of the wells contain more than one primer pair. Following PCR, the microtiter plate is placed on a fully automated instrument and ESI-MS analysis is performed. The mass spectrometer is an analytical tool that effectively weighs the amplicons, or mixture of amplicons, with sufficient mass accuracy that the composition of A, G, C, and T can be deduced for each amplicon in the PCR reaction. The determined composition is then interrogated using a proprietary software database.

The above two platforms allow for direct detection of the nucleic acid of microbes directly from clinical samples without a culture step because they use a PCR amplification reaction. Another application is using MALDI-TOF to identify bacteria and yeast isolates recovered in clinical cultures. These platforms target the highly abundant ribosomal proteins of bacteria and yeast. The assays involve making a thin smear from a colony or broth culture onto a metallic slide of the organism to be identified and applying an acid matrix to it. The slide is placed into the instrument where the organism mixture is hit by laser pulses. Charged protein fragments are produced and accelerated through an electrostatic field in a vacuum tube until they contact the mass spectrometer’s detector. Molecules of different masses and charges “fly” at different speeds (“time of flight”). A spectral signature, generally in the range of 1000–20,000 mass-to-charge ratio (m/z), is generated. This spectral signal is compared to others in the proprietary databases of each instrument for genus or species assignment of the organism. The reader is referred to the Patel reference for additional details.

Organisms such as M tuberculosis, Salmonella serovar Typhi, and Brucella species are considered pathogens whenever they are found in patient specimens. However, many infections are caused by organisms that are permanent or transient members of the normal microbiota. For example, Escherichia coli is part of the normal gastrointestinal microbiota, but is also the most common cause of urinary tract infections. Similarly, the vast majority of mixed bacterial infections with anaerobes are caused by organisms that are members of the normal microbiota.

The relative numbers of specific organisms found in a culture are important when members of the normal microbiota are the cause of infection. When numerous gram- negative rods of species such as Klebsiella pneumoniae are found mixed with a few normal nasopharyngeal bacteria in a sputum culture, the gram-negative rods are strongly suspect as the cause of pneumonia because large numbers of gram-negative rods are not normally found in sputum or in the nasopharyngeal microbiota; the organisms should be identified and reported. In contrast, abdominal abscesses commonly contain a normal distribution of aerobic, facultatively anaerobic, and obligately anaerobic organisms, representative of the gastrointestinal microbiota. In such cases, identification of all species present is not warranted; instead, it is appropriate to report “normal gastrointestinal microbiota.”

Yeasts in small numbers are commonly part of the normal microbial microbiota. However, other fungi are not normally present and therefore should be identified and reported. Viruses usually are not part of the normal microbiota as detected in diagnostic microbiology laboratories, but can be found in otherwise healthy individuals, presumably as asymptomatic infections. Latent viruses such as herpes simplex or CMV, or live vaccine viruses such as poliovirus can be detected in asymptomatic cases. In some parts of the world, stool specimens commonly yield evidence of parasitic infection without symptoms present. Therefore, the clinical presentation of infectious disease illness along with the relative number of potentially pathogenic organisms is important in establishing the correct diagnosis.

Members of the normal microbiota that are most commonly present in patient specimens and that may be reported as “normal microbiota” are discussed in Chapter 10.

The antimicrobial drug used initially in the treatment of an infection is chosen on the basis of clinical impression after the clinician is convinced that an infection exists and has made a tentative etiologic diagnosis on clinical grounds. On the basis of this “best guess,” a drug that is likely to be effective against the suspect agent(s) can be selected (see Chapter 28). Before this drug is administered, specimens are obtained for laboratory detection of the causative agent. The results of these examinations may allow for narrowing of antibiotics to targeted therapy (as opposed to broad gram-positive and gram-negative coverage for sepsis). The identification of certain microorganisms that are uniformly drug-susceptible eliminates the necessity for further testing and permits the selection of optimally effective drugs based on the organism’s known susceptibility profile. When the organism resistance profile is varied, tests for drug susceptibility of isolated microorganisms will guide optimal drug choice (see Chapter 28). Disk diffusion susceptibility tests measure the ability of bacteria to grow on the surface of an agar plate in the presence of paper disks containing antibiotic drug. The drug diffuses out into the surrounding agar, inhibiting bacterial growth in a circular area surrounding the disk. The diameter of this zone of growth inhibition is measured, and correlates with the susceptibility of the isolate being tested. The choice of drugs to be included in a routine susceptibility test battery should be based on the susceptibility patterns of isolates in the laboratory, the type of infection (community-acquired or nosocomial), the source of the infection, and cost-effectiveness analysis for the patient population. The Clinical and Laboratory Standards Institute (CLSI) (Wayne, PA) provides recommendations for which agents to test based on the organism recovered and the specimen type, and interpretive criteria (susceptible, intermediate, or resistant) based on the measured zone size.

The sizes of zones of growth inhibition vary with the pharmacologic characteristics of different drugs. Thus, the zone size of one drug cannot be compared to the zone size of another drug acting on the same organism. However, for any one drug the zone size can be compared to a standard, provided that media, inoculum size, and other conditions are carefully controlled. This makes it possible to define for each drug a diameter of inhibition zone that distinguishes susceptible from intermediate or resistant strains.

The disk test measures the ability of drugs to inhibit the growth of bacteria in vitro. The results correlate reasonably well with therapeutic response in those disease processes in vivo when body defenses can eliminate infectious microorganisms, but may be less well correlated with response in immunocompromised patients. The selection of appropriate antibiotic therapy depends on clinical as well as bacterial factors, such as use of bactericidal rather than bacteriostatic drugs for endocarditis, or drugs that will penetrate the blood–brain barrier for central nervous system infections (see Chapter 28). Minimum inhibitory concentration (MIC) tests measure the ability of organism to grow in broth culture in the presence of various dilutions of antibiotics. It measures more exactly the concentration of an antibiotic necessary to inhibit growth of a standardized inoculum under defined conditions. A semiautomated microdilution method is used in which defined amounts of drug are dissolved in a measured small volume of broth and inoculated with a standardized number of microorganisms. The end point, or MIC, is considered the last broth cup (lowest concentration of drug) remaining clear, ie, free from microbial growth. The MIC provides a better estimate of the probable amount of drug necessary to inhibit growth in vivo and thus helps in gauging the dosage regimen necessary for the patient. Guidelines available from CLSI provide interpretive criteria, defining strains as resistant, intermediate, or susceptible to a certain drug based on the MIC.

The MIC only shows that bacterial growth is inhibited at that drug concentration; there may still be viable bacteria that can recover when the drug is removed. Bactericidal effects can be estimated by subculturing the clear broth from MIC testing onto antibiotic-free solid media. The result, eg, a reduction of colony-forming units by 99.9% below that of the control, is called the minimal bactericidal concentration (MBC).

Because empiric therapy must often be given before the results of antimicrobial susceptibility tests are available, it is recommended by CLSI that laboratories publish an antibiogram annually that contains the results of susceptibility testing in aggregate for particular organism–drug combinations. For example, it may be important to know the most active β-lactam antimicrobial agent targeted against Pseudomonas aeruginosa among intensive care unit patients in a particular hospital. This allows the best therapy to be chosen based on clinical suspicion of the infecting organism and known locally circulating strains.

The selection of a bactericidal drug or drug combination for each patient can be guided by specialized laboratory tests. Such tests measure either the rate of killing (time-kill assay) or the proportion of the microbial population that is killed in a fixed time by patient serum (serum bactericidal testing). Synergy testing measures the ability of drugs to enhance bacterial killing when present in combination; drugs showing synergy may be more effective when given together to treat infection. Few clinical laboratories perform this type of specialized susceptibility testing.

Wounds, Tissues, Bones, Abscesses, and Fluids

Microscopic study of smears and culture of specimens from wounds or abscesses often gives early and important indications of the nature of the infecting organism and thus helps in the choice of antimicrobial drugs. Specimens from diagnostic tissue biopsies should be submitted for microbiologic as well as histologic examination. Such specimens for bacteriologic examination are submitted fresh, without fixatives or disinfectants, and are cultured by a variety of methods.

The pus in closed, undrained soft tissue abscesses frequently contains only one organism as the infecting agent; most commonly staphylococci, streptococci, or enteric gram-negative rods. The same is true in acute osteomyelitis, where the organisms can often be cultured from blood before the infection has become chronic. Multiple microorganisms are frequently encountered in abdominal abscesses and abscesses contiguous with mucosal surfaces as well as in open wounds. When deep suppurating lesions, such as chronic osteomyelitis, drain onto exterior surfaces through a sinus or fistula, the microbiota of the surface through which the lesion drains must not be mistaken for that of the deep lesion. Instead, specimens should be aspirated from the primary infection through uninfected tissue.

Bacteriologic examination of pus from closed or deep lesions must include culture by anaerobic methods. Anaerobic bacteria (Bacteroides, Fusobacteria, etc) sometimes play an essential causative role, and mixtures of aerobes and anaerobes are often present.

The methods used for cultures must be suitable for the semiquantitative recovery of common bacteria and also for recovery of specialized microorganisms, including mycobacteria and fungi. Eroded skin and mucous membranes are frequently the sites of yeast or fungus infections. Candida, Aspergillus, and other yeasts or fungi can be seen microscopically in smears or scrapings from suspicious areas and can be grown in cultures. Treatment of a specimen with KOH and calcofluor white greatly enhances the observation of yeasts and molds in the specimen.

Exudates that have collected in the pleural, peritoneal, pericardial, or synovial spaces must be aspirated with aseptic technique. If the material is frankly purulent, smears and cultures are made directly. If the fluid is clear, it can be centrifuged and the sediment used for stained smears and cultures. The culture method used must be suitable for the growth of organisms suspected on clinical grounds—for example, mycobacteria, anaerobic organisms—as well as the commonly encountered pyogenic bacteria. Some fluid specimens clot, and culture of an anticoagulated specimen may be necessary. The following chemistry and hematology results are suggestive of infection: specific gravity greater than 1.018, protein content greater than 3 g/dL (often resulting in clotting), and white cell counts greater than 500–1000/μL. Polymorphonuclear leukocytes (PMNs) predominate in acute untreated pyogenic infections; lymphocytes or monocytes predominate in chronic infections. Transudates resulting from neoplastic growth may grossly resemble infectious exudates by appearing bloody or purulent and by clotting on standing. Cytologic study of smears or of sections of centrifuged cells may demonstrate the neoplastic nature of the process.

Blood

Since bacteremia frequently portends life-threatening illness, its early detection is essential. Blood culture is the single most important procedure to detect systemic infection due to bacteria. It provides valuable information for the management of febrile, acutely ill patients with or without localizing symptoms and signs and is essential in any patient in whom infective endocarditis is suspected even if the patient does not appear acutely or severely ill. In addition to its diagnostic significance, recovery of an infectious agent from the blood provides invaluable aid in determining antimicrobial therapy. Every effort should therefore be made to isolate the causative organisms in bacteremia.

In healthy persons, properly obtained blood specimens are sterile. Although microorganisms from the normal respiratory and gastrointestinal microbiota occasionally enter the blood, they are rapidly removed by the reticuloendothelial system. These transients rarely affect the interpretation of blood culture results. If a blood culture yields microorganisms, this fact is of great clinical significance provided that contamination can be excluded. Contamination of blood cultures with normal skin microbiota is most commonly due to errors in the blood collection procedure. Therefore, proper technique in performing a blood culture is essential.

The following rules, rigidly applied, yield reliable results:

1. Use strict aseptic technique. Wear gloves—they do not have to be sterile.

2. Apply a tourniquet and locate a fixed vein by touch. Release the tourniquet while the skin is being prepared.

3. Prepare the skin for venipuncture by cleansing it vigorously with 70–95% isopropyl alcohol. Using 2% tincture of iodine or 2% chlorhexidine, start at the venipuncture site and cleanse the skin in concentric circles of increasing diameter. Allow the antiseptic preparation to dry for at least 30 seconds. Do not touch the skin after it has been prepared.

4. Reapply the tourniquet, perform venipuncture, and (for adults) withdraw approximately 20 mL of blood.

5. Add the blood to aerobic and anaerobic blood culture bottles.

6. Properly label and promptly transport the specimens to the laboratory.

Several factors determine whether blood cultures will yield positive results: the volume of blood cultured, the dilution of blood in the culture medium, the use of both aerobic and anaerobic culture media, and the duration of incubation. For adults, 20 mL per culture is usually obtained, and half is placed in an aerobic blood culture bottle and half in an anaerobic one, with one pair of bottles comprising a single blood culture. Commercial manufacturers of blood culture systems optimize the broth composition, volume, and antibiotic neutralizing agents used (activated charcoal or resin beads). Automated blood culture systems use a variety of methods to detect positive cultures. These automated methods allow frequent monitoring of the cultures—as often as every few minutes—and earlier detection of positive ones. The media in the automated blood culture systems are so enriched and the detection systems so sensitive that blood cultures using the automated systems do not need to be processed for more than 5 days. In general, subcultures are indicated only when the machine indicates that the culture is positive. Manual blood culture systems are obsolete and are likely to be used only in laboratories in developing countries that lack the resources to purchase automated blood culturing systems. In manual systems, the blood culture bottles are examined two or three times a day for the first 2 days and daily thereafter for 1 week. In the manual method, blind subcultures of all the blood culture bottles on days 2 and 7 may be necessary.

The number of blood specimens that should be drawn for cultures and the period of time over which this is done depend in part on the severity of the clinical illness. In hyperacute infections, eg, gram-negative sepsis with shock or staphylococcal sepsis, it is appropriate to obtain a minimum of two blood cultures from different anatomic sites, preferably through peripheral venipuncture. More recent literature has suggested that three to four blood cultures may be necessary. In other bacteremic infections, eg, subacute endocarditis, three blood specimens should be obtained over 24 hours. A total of three blood cultures yields the infecting bacteria in more than 95% of bacteremic patients. If the initial three cultures are negative and occult abscess, fever of unexplained origin, or some other obscure infection is suspected, additional blood specimens should be cultured when possible before antimicrobial therapy is started.

It is necessary to determine the significance of a positive blood culture. The following criteria may be helpful in differentiating “true positives” from contaminated specimens:

1. Growth of the same organism in repeated cultures obtained at different times from separate anatomic sites strongly suggests true bacteremia.

2. Growth of different organisms in different culture bottles suggests contamination but occasionally may follow clinical problems such as wound sepsis or ruptured bowel.

3. Growth of normal skin microbiota, eg, coagulase-negative staphylococci, diphtheroids (corynebacteria and propionibacteria), or anaerobic gram-positive cocci, in only one of several cultures suggests contamination. Growth of such organisms in more than one culture or from specimens from a high-risk patient, such as an immunocompromised bone marrow transplant recipient, enhances the likelihood that clinically significant bacteremia exists.

4. Organisms such as viridans streptococci or enterococci are likely to grow in blood cultures from patients suspected to have endocarditis, and gram-negative rods such as E coli are likely to grow in blood cultures from patients with clinical gram-negative sepsis. Therefore, when such “expected” organisms are found, they are more apt to be etiologically significant.

The following are the bacterial species most commonly recovered in positive blood cultures: staphylococci, including S aureus; viridans streptococci; enterococci, including Enterococcus faecalis; gram-negative enteric bacteria, including E coli and K pneumoniae; P aeruginosa; pneumococci; and Haemophilus influenzae. Candida species, other yeasts, and some dimorphic fungi such as H capsulatum grow in blood cultures, but many fungi are rarely, if ever, isolated from blood. CMV and HSV can occasionally be cultured from blood, but most viruses and rickettsiae and chlamydiae are not cultured from blood. Parasitic protozoa and helminths do not grow in blood cultures.

In most types of bacteremia, examination of direct blood smears is not useful. Diligent examination of Gram-stained smears of the buffy coat from anticoagulated blood will occasionally show bacteria in patients with S aureus infection, clostridial sepsis, or relapsing fever. In some microbial infections (eg, anthrax, plague, relapsing fever, rickettsiosis, leptospirosis, spirillosis, psittacosis), inoculation of blood into animals may give positive results more readily than does culture. In practicality, this is never done in clinical laboratories and diagnosis may be made by alternate means such as serology or nucleic acid amplification tests.

Urine

Bacteriologic examination of the urine is done mainly when signs or symptoms point to urinary tract infection, renal insufficiency, or hypertension. It should always be done in persons with suspected systemic infection or fever of unknown origin. It is desirable for women in the first trimester of pregnancy to be assessed for asymptomatic bacteriuria.

Urine secreted in the kidney is sterile unless the kidney is infected. Uncontaminated bladder urine is also normally sterile. The urethra, however, contains a normal microbiota, so that normal voided urine contains small numbers of bacteria. Because it is necessary to distinguish contaminating organisms from etiologically important organisms, only quantitative urine examination can yield meaningful results.

The following steps are essential in proper urine examination.

A. Proper Collection of Specimen

Proper collection of the specimen is the single most important step in a urine culture and the most difficult. Satisfactory specimens from females are problematic.

1. Have at hand a sterile, screw-cap specimen container and two to three gauze sponges soaked with nonbacteriostatic saline (antibacterial soaps for cleansing are not recommended).

2. Spread the labia with two fingers and keep them spread during the cleansing and collection process. Wipe the urethra area once from front to back with each of the saline gauzes.

3. Start the urine stream and, using the urine cup, collect a midstream specimen. Properly label the cup.

The same method is used to collect specimens from males; the foreskin should be kept retracted in uncircumcised males.

Catheterization carries a risk of introducing microorganisms into the bladder, but it is sometimes unavoidable. Separate specimens from the right and left kidneys and ureters can be obtained by the urologist using a catheter at cystoscopy. When an indwelling catheter and closed collection system are in place, urine should be obtained by sterile aspiration of the catheter with needle and syringe, not from the collection bag. To resolve diagnostic problems, urine can be aspirated aseptically directly from the full bladder by means of suprapubic puncture of the abdominal wall. This procedure is usually done in infants.

For most examinations, 0.5 mL of ureteral urine or 5 mL of voided urine is sufficient. Because many types of microorganisms multiply rapidly in urine at room or body temperature, urine specimens must be delivered to the laboratory rapidly or refrigerated not longer than overnight. Alternatively, transport tubes that contain boric acid may be used if specimens cannot be refrigerated.

B. Microscopic Examination

Much can be learned from simple microscopic examination of urine. A drop of fresh uncentrifuged urine placed on a slide, covered with a coverglass, and examined with restricted light intensity under the high-dry objective of an ordinary clinical microscope can reveal leukocytes, epithelial cells, and bacteria if more than 10⁵/mL are present. Finding at least 10⁵ organisms per milliliter in a properly collected and examined urine specimen is strong evidence of active urinary tract infection. A Gram-stained smear of uncentrifuged midstream urine that shows gram-negative rods is diagnostic of a urinary tract infection.

Brief centrifugation of urine readily sediments pus cells, which may carry along bacteria and thus may help in microscopic diagnosis of infection. The presence of other formed elements in the sediments—or the presence of proteinuria—is of little direct aid in the specific identification of active urinary tract infection. Pus cells may be present without bacteria, and, conversely, bacteriuria may be present without pyuria. The presence of many squamous epithelial cells, lactobacilli, or mixed flora on culture suggests improper urine collection.

Some urine dipsticks contain leukocyte esterase and nitrite, measurements of polymorphonuclear cells and bacteria, respectively, in the urine. Positive reactions are strongly suggestive of bacterial urinary tract infection, while negative reactions for both indicate a low likelihood of urinary tract infection, except for neonates and immunocompromised patients.

C. Culture

Culture of the urine, to be meaningful, must be performed quantitatively. Properly collected urine is cultured in measured amounts on solid media, and the colonies that appear after incubation are counted to indicate the number of bacteria per milliliter. The usual procedure is to spread 0.001–0.05 mL of undiluted urine on blood agar plates and other solid media for quantitative culture. All media are incubated overnight at 37°C; growth density is then compared with photographs of different densities of growth for similar bacteria, yielding semiquantitative data.

In active pyelonephritis, the number of bacteria in urine collected by ureteral catheter is relatively low. While accumulating in the bladder, bacteria multiply rapidly and soon reach numbers in excess of 10⁵/mL—far more than could occur as a result of contamination by urethral or skin microbiota or from the air. Therefore, it is generally agreed that if more than 10⁵ colonies/mL are cultivated from a properly collected and properly cultured urine specimen, this constitutes strong evidence of active urinary tract infection. The presence of 10⁵ bacteria or more of the same type per milliliter in two consecutive specimens establishes a diagnosis of active infection of the urinary tract with 95% certainty. If fewer bacteria are cultivated, repeated examination of urine is indicated to establish the presence of infection.

The presence of fewer than 10⁴ bacteria per milliliter, including several different types of bacteria, suggests that organisms come from the normal microbiota and are contaminants, usually from an improperly collected specimen. The presence of 10⁴/mL of a single type of enteric gram-negative rod is strongly suggestive of urinary tract infection, especially in men. Occasionally, young women with acute dysuria and urinary tract infection will have 10²–10³/mL. If cultures are negative but clinical signs of urinary tract infection are present, “urethral syndrome,” ureteral obstruction, tuberculosis of the bladder, gonococcal infection, or other disease must be considered.

Cerebrospinal Fluid

Meningitis ranks high among medical emergencies, and early, rapid, and precise diagnosis is essential. Diagnosis of meningitis depends on maintaining a high index of suspicion, securing adequate specimens properly, and examining the specimens promptly. Because the risk of death or irreversible damage is great unless treatment is started immediately, there is rarely a second chance to obtain pretreatment specimens, which are essential for specific etiologic diagnosis and optimal management.

The most urgent diagnostic issue is the differentiation of acute purulent bacterial meningitis from “aseptic” and granulomatous meningitis. The immediate decision is usually based on the cell count, the glucose concentration and protein content of CSF, and the results of microscopic search for microorganisms (see Case 1, Chapter 48). The initial impression is modified by the results of culture, serologic tests, nucleic acid amplification tests, and other laboratory procedures. In evaluating the results of CSF glucose determinations, the simultaneous blood glucose level must be considered. In some central nervous system neoplasms, the CSF glucose level is low.

A. Specimens

As soon as infection of the central nervous system is suspected, blood samples are taken for culture, and CSF is obtained. To obtain CSF, perform lumbar puncture with strict aseptic technique, taking care not to risk compression of the medulla by too rapid withdrawal of fluid when the intracranial pressure is markedly elevated. CSF is usually collected in three to four portions of 2–5 mL each, in sterile tubes. This permits the most convenient and reliable performance of tests to determine the several different values needed to plan a course of action.

B. Microscopic Examination

Smears are made from the sediment of centrifuged CSF. Using a cytospin centrifuge to prepare the slides for staining is recommended because it concentrates cellular material and bacterial cells more effectively than standard centrifugation. Smears are stained with Gram stain. Study of stained smears under the oil immersion objective may reveal intracellular gram-negative diplococci (meningococci), intra- and extracellular lancet-shaped gram-positive diplococci (pneumococci), or small gram-negative rods (H influenzae or enteric gram-negative rods).

C. Antigen Detection

Cryptococcal antigen in CSF may be detected by a latex agglutination or EIA test. Bacterial antigen detection tests have been developed but have fallen out of favor as they are not more sensitive than routine Gram stain.

D. Culture

The culture methods used must favor the growth of microorganisms most commonly encountered in meningitis. Sheep blood and chocolate agar together grow almost all bacteria and fungi that cause meningitis. The diagnosis of tuberculous meningitis requires cultures on special media (see Table 47-2 and Chapter 23). Viruses causing aseptic meningitis or meningoencephalitis, such as herpes simplex, enterovirus, JC virus, and mumps, can be best detected by nucleic acid amplification methods.

E. Follow-Up Examination of Cerebrospinal Fluid

The return of the CSF glucose level and cell count toward normal is good evidence of adequate therapy. The clinical response is of paramount importance.

Respiratory Secretions

Symptoms or signs often point to involvement of a particular part of the respiratory tract, and specimens are chosen accordingly. In interpreting laboratory results, it is necessary to consider the normal microbiota of the area from which the specimen was collected.

A. Specimens

1. Throat—Most “sore throats” are due to viral infection. Only 5–10% of “sore throats” in adults and 15–20% in children are associated with bacterial infections. The finding of a follicular yellowish exudate or a grayish membrane must arouse the suspicion that Lance-field group A β-hemolytic streptococcal, diphtherial, gonococcal, fusospirochetal, or candidal infection exists; such signs may also be present in infectious mononucleosis, adenovirus, and other virus infections.

Throat swabs are taken from each tonsillar area and from the posterior pharyngeal wall without touching the tongue or the buccal mucosa. The normal throat microbiota includes an abundance of viridans streptococci, neisseriae, diphtheroids, staphylococci, small gram-negative rods, and many other organisms. Microscopic examination of smears from throat swabs is of little value in streptococcal infections, because all throats harbor a predominance of streptococci.

Cultures of throat swabs are most reliable if inoculated promptly after collection. Media selective for streptococci can be used to culture for group A streptococci. In streaking selective media for streptococci or blood agar culture plates, it is essential to spread a small inoculum thoroughly and avoid overgrowth by normal microbiota. This can be done readily by touching the throat swab to one small area of the plate and using a second, sterile applicator (or sterile bacteriologic loop) to streak the plate from that area. Detection of β-hemolytic colonies is facilitated by slashing the agar (to provide reduced oxygen tension) and incubating the plate for 2 days at 37°C.

Over the last two decades, a variety of antigen detection tests, probe methods, and nucleic acid amplification tests have been developed to enhance the detection of Streptococcus pyogenes from throat swabs in patients with acute streptococcal pharyngitis. It is important that the user realizes that only S pyogenes will be detected or excluded by these tests, and thus, they cannot be relied on to diagnose bacterial pharyngitis caused by other pathogens. Current recommendations indicate performing culture on certain patients with suspected group A streptococcal throat infections, particularly in the pediatric setting, who have negative rapid test results, unless the rapid tests have been shown to be as sensitive as culture methods.

2. Nasopharynx—Nasopharyngeal swabs are most commonly used in the diagnosis of respiratory viral infections. Whooping cough is diagnosed by culture of B pertussis from nasopharyngeal or nasal washings or by PCR amplification of B pertussis DNA in the specimen.

3. Middle ear—Specimens are rarely obtained from the middle ear because puncture of the tympanic membrane is necessary. In acute otitis media, 30–50% of aspirated fluids are bacteriologically sterile. The most frequently isolated bacteria are pneumococci, H influenzae, Moraxella catarrhalis, and hemolytic streptococci.

4. Lower respiratory tract—Bronchial and pulmonary secretions of exudates are often studied by examining sputum. The most misleading aspect of sputum examination is the almost inevitable contamination with saliva and mouth microbiota. Thus, finding candida, S aureus, or even S pneumoniae in the sputum of a patient with pneumonitis has no etiologic significance unless supported by the clinical picture. Meaningful sputum specimens should be expectorated from the lower respiratory tract and should be grossly distinct from saliva. Sputum may be induced by the inhalation of heated hypertonic saline aerosol for several minutes. In pneumonia accompanied by a pleural effusion, the pleural fluid may yield the causative organisms more reliably than does sputum. In suspected tuberculosis, gastric washings (swallowed sputum) may yield organisms when expectorated material is not obtainable, eg, in the pediatric patient.

5. Transtracheal aspiration, bronchoscopy, lung biopsy, bronchoalveolar lavage—The microbiota in such specimens often reflects accurately the events in the lower respiratory tract. Specimens obtained by bronchoscopy may be necessary in the diagnosis of Pneumocystis pneumonia or infection due to Legionella or other organisms. Bronchoalveolar lavage specimens are particularly useful in immunocompromised patients with diffuse pneumonia.

B. Microscopic Examination

Smears of purulent flecks or granules from sputum stained by Gram stain or acid-fast methods may reveal causative organisms and PMNs. The presence of many squamous epithelial cells suggests heavy contamination with saliva and such samples will be rejected for culture; a large number of PMNs suggests a purulent exudate from infection.

C. Culture

The media used for sputum cultures must be suitable for the growth of bacteria (eg, pneumococci, Klebsiella), fungi (eg, C immitis), mycobacteria (eg, M tuberculosis), and other organisms. Specimens obtained by bronchoscopy and lung biopsy should also be cultured on other media (eg, for anaerobes, Legionella, and others). The relative prevalence of different organisms in the specimen must be estimated. Only a finding of one predominant organism or the simultaneous isolation of an organism from both sputum and blood can clearly establish its role in a pneumonic or suppurative process. Laboratories in hospitals that have large transplant populations often have comprehensive algorithms for specimens that are obtained by bronchoscope that include a variety of diagnostic methods including NAATs and other techniques for broad pathogen detection.

Gastrointestinal Tract Specimens

Acute symptoms referable to the gastrointestinal tract, particularly nausea, vomiting, and diarrhea, are commonly attributed to infection. In reality, most such attacks are caused by intolerance to food or drink, enterotoxins, drugs, or systemic illnesses.

Many cases of acute infectious diarrhea are due to viruses, which cannot be grown in tissue culture. On the other hand, many viruses that can be grown in culture (eg, adenoviruses, enteroviruses) can multiply in the gut without causing gastrointestinal symptoms. Similarly, some enteric bacterial pathogens may persist in the gut following an acute infection. Thus, it may be difficult to assign significance to a bacterial or viral agent cultured from the stool, especially in subacute or chronic illness.

These considerations should not discourage the physician from attempting laboratory isolation of enteric organisms but should constitute a warning of some common difficulties in interpreting the results.

The lower bowel has an exceedingly large normal bacterial microbiota. The most prevalent organisms are anaerobes (Bacteroides, gram-positive rods, and gram-positive cocci), gram-negative enteric organisms, and E faecalis. Any attempt to recover pathogenic bacteria from feces involves separation of pathogens from the normal microbiota, usually through the use of differential selective media and enrichment cultures. Important causes of acute gastroenteritis include viruses, toxins (of staphylococci, clostridia, vibrios, toxigenic E coli), shigellae and salmonellae, and campylobacters. The relative importance of these groups of organisms differs greatly in various parts of the world.

A. Specimens

Feces and rectal swabs are the most readily available specimens. Bile obtained by duodenal drainage may reveal infection of the biliary tract. The presence of blood, mucus, or helminths must be noted on gross inspection of the specimen. Leukocytes seen in suspensions of stool examined microscopically or detection of the leukocyte-derived protein lactoferrin are useful means of differentiating inflammatory from noninflammatory diarrhea, but do not distinguish infection from noninfectious gastrointestinal conditions. Special techniques must be used to search for parasitic protozoa and helminths and their ova.

B. Culture

Specimens are suspended in broth and cultured on ordinary as well as differential media (eg, MacConkey agar, EMB agar) to permit separation of non–lactose-fermenting gram-negative rods from other enteric bacteria. If salmonella infection is suspected, the specimen is also placed in an enrichment medium (eg, selenite F broth) for 18 hours before being plated on differential media (eg, Hektoen enteric or Shigella-Salmonella agar). Yersinia enterocolitica is more likely to be isolated after storage of fecal suspensions for 2 weeks at 4°C, but it can be isolated on yersinia or Shigella-Salmonella agar incubated at 25°C. Vibrios grow best on thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Thermophilic campylobacters are isolated on Campy lobacter agar or Skirrow’s selective medium incubated at 40–42°C in 10% CO₂ with greatly reduced O₂ tension. Bacterial colonies are identified by standard bacteriologic methods or MS. Agglutination of bacteria from suspect colonies by pooled specific antiserum is often the fastest way to establish the presence of salmonellae or shigellae in the intestinal tract.

C. Non–Culture-Based Methods

EIAs for detection of specific enteric pathogens, either directly in stool specimens or to confirm growth in broth or on plated media, are available. EIAs that detect Shiga toxins 1 and 2 in suspected cases of colitis caused by enterohemorrhagic E coli (also called Shiga toxin–producing E coli or STEC) are available and are superior to culture. Also available are EIAs for direct detection of viral pathogens such as rotavirus, adenoviruses 40 and 41, and noroviruses; bacterial pathogens such as Campylobacter jejuni; and the protozoan parasites Giardia lamblia, Cryptosporidium parvum, and Entamoeba histolytica. The performance of these assays is variable. Nucleic acid testing panels are available for the direct detection of gastrointestinal pathogens in stool. Specimen requirements and the organisms present on each panel vary by manufacturer.

Intestinal parasites and their ova are identified by microscopic study of fresh fecal specimens. The specimens require special handling in the laboratory and multiple specimens may be needed to diagnose low-level infections (see Chapter 46). Nucleic acid testing can be used to detect some parasites.

Sexually Transmitted Diseases

The causes of the genital discharge of urethritis in men are N gonorrhoeae, C trachomatis, and Ureaplasma urealyticum. Endocervicitis in women is caused by N gonorrhoeae and C trachomatis. The genital sores associated with diseases in both men and women are often caused by HSV, less commonly T pallidum (syphilis) or Haemophilus ducreyi (chancroid), uncommonly lymphogranuloma venereum serovars of C trachomatis, and rarely Klebsiella granulomatis (granuloma inguinale). Each of these diseases has a characteristic natural history and evolution of lesions, but one can mimic another. The laboratory diagnosis of most of these infections is covered elsewhere in this book. A few diagnostic tests are listed below and outlined in Table 47-2.

A. Gonorrhea

A stained smear of a urethral or a cervical exudate that shows intracellular gram-negative diplococci strongly suggests gonorrhea. The sensitivity is about 90% for men and 50% for women—thus, culture or a nucleic acid amplification test is recommended for women. Exudate, rectal swab, or throat swab must be plated promptly on special media to yield N gonorrhoeae. Molecular methods to detect N gonorrhoeae DNA in urethral or cervical exudates or urine are more sensitive than culture.

B. Chlamydial Genital Infections

See later section in this chapter on the diagnosis of chlamydial infections.

C. Genital Herpes

See Chapter 33 and the later section in this chapter on the diagnosis of viral infections.

D. Syphilis

Dark-field or immunofluorescence examination of fresh tissue fluid expressed from the base of the chancre may reveal typical T pallidum, but this testing is rarely clinically available. Serologic tests for syphilis become positive 3–6 weeks after infection. A positive nontreponemal test (eg, VDRL or RPR) requires confirmation. A positive immunofluorescent treponemal antibody test (eg, FTA-ABS, T pallidum particle agglutination [TP-PA], or the newer Treponema EIAs and chemiluminescence assays—see Chapter 24) proves syphilitic infection.

E. Chancroid

Smears from a suppurating lesion usually show a mixed bacterial flora. Swabs from lesions should be cultured at 33°C on two or three media that are selective for H ducreyi. Serologic tests are not helpful. Culture is only about 50% sensitive, so diagnosis and treatment are often made empirically based on a typical presentation. Molecular assays are used in some reference or research laboratories.

F. Granuloma Inguinale

Klebsiella (formerly Calymmatobacterium) granulomatis, the causative agent of this hard, granulomatous, proliferating lesion, can be grown in complex bacteriologic media, but this is rarely attempted and very difficult to perform successfully. Histologic demonstration of intracellular “Donovan bodies” in biopsy material most frequently supports the clinical impression. Serologic tests are not helpful. Molecular assays are used in some reference or research laboratories.

G. Vaginosis/Vaginitis

Bacterial vaginosis associated with Gardnerella vaginalis or Mobiluncus (see Chapter 21 and Case 13, Chapter 48) is diagnosed in the examining room by inspection of the vaginal discharge; the discharge (1) is grayish and sometimes frothy, (2) has a pH above 4.6, (3) has an amine (“fishy”) odor when alkalinized with potassium hydroxide, and (4) contains “clue cells,” large epithelial cells covered with gram-negative or gram-variable rods. Similar observations are used to diagnose Trichomonas vaginalis (see Chapter 46) infection; the motile organisms can be seen in wet-mount preparations or cultured from genital discharge. Trichomonas culture, DNA probes, and NAATs are much more sensitive than wet-mount procedures. Candida albicans vaginitis is diagnosed by finding yeast or pseudohyphae in a potassium hydroxide preparation of the vaginal discharge, by probes, or by culture.

A large majority of the bacteria that make up the normal human microbiota are anaerobes. When displaced from their normal sites into tissues or body spaces, anaerobes may produce disease. Certain characteristics are suggestive of anaerobic infections: (1) They are often contiguous with a mucosal surface. (2) They tend to involve mixtures of organisms. (3) They tend to form closed-space infections, either as discrete abscesses (lung, brain, pleura, peritoneum, pelvis) or by burrowing through tissue layers. (4) Pus from anaerobic infections often has a foul odor. (5) Most of the pathogenically important anaerobes except Bacteroides and some Prevotella species are highly susceptible to penicillin G. (6) Anaerobic infections are favored by reduced blood supply, necrotic tissue, and a low oxidation-reduction potential, all of which also interfere with delivery of antimicrobial drugs. (7) It is essential to use special collection methods, transport media, and sensitive anaerobic techniques and media to isolate the organisms. Otherwise, bacteriologic examination may be negative or yield only incidental aerobes. (See also Chapter 21.)

The following are sites of important anaerobic infections.

Respiratory Tract

Periodontal infections, perioral abscesses, sinusitis, and mastoiditis may involve predominantly Prevotella melaninogenica, Fusobacterium, and peptostreptococci. Aspiration of oral cavity contents into the lung may result in necrotizing pneumonia, lung abscess, and empyema. Antimicrobial drugs and postural or surgical drainage are essential for treatment.

Central Nervous System

Anaerobes rarely produce meningitis but are common causes of brain abscess, subdural empyema, and septic thrombophlebitis. The organisms usually originate in the respiratory tract and spread to the brain via extension or hematogenously.

Intra-Abdominal and Pelvic Infections

The microbiota of the colon consists predominantly of anaerobes, 10¹¹ per gram of feces. Bacteroides fragilis, clostridia, and peptostreptococci play a main role in abscess formation originating in perforation of the bowel. Prevotella bivia and Prevotella disiens are important in abscesses of the pelvis originating in the female genital organs. Like B fragilis, these species are often relatively resistant to penicillin; therefore, clindamycin, metronidazole, or another effective agent should be used.

Skin and Soft Tissue Infections

Anaerobes and aerobic bacteria often join to form synergistic infections (gangrene, necrotizing fasciitis, cellulitis). Surgical drainage, excision, and improved circulation are the most important forms of treatment, while antimicrobial drugs act as adjuncts. It is usually difficult to pinpoint one specific organism as being responsible for the progressive lesion, since mixtures of organisms are usually involved.

Although C trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci are bacteria, they are obligate intracellular parasites. Cultures and other diagnostic tests for chlamydia require procedures much like those used in diagnostic virology laboratories rather than those used in bacteriology and mycology laboratories. Thus, the diagnosis of chlamydial infections is discussed in a separate section of this chapter. The laboratory diagnosis of chlamydial infections also is discussed in Chapter 27.

Specimens

For C trachomatis ocular and genital infections, the specimens for direct examination or culture must be collected from infected sites by vigorous swabbing or scraping of the involved epithelial cell surface. Cultures of purulent discharges are not adequate, and purulent material should be cleaned away before the specimen is obtained. Thus, for inclusion conjunctivitis, a conjunctival scraping is obtained; for urethritis, a swab specimen is obtained from several centimeters into the urethra; and for cervicitis, a specimen is obtained from the columnar cell surface of the endocervical canal. Swab or urine samples may be used for nucleic acid amplification tests. When upper genital tract infection is suspected in women, scrapings of the endometrium provide a good sample. Fluid obtained by culdocentesis or aspiration of the uterine tube has a low yield for C trachomatis on culture.

For C pneumoniae, use nasopharyngeal (not throat) swab specimens.

For lymphogranuloma venereum, aspirates of buboes or fluctuant nodes provide the best specimen for culture.

For psittacosis, culture of sputum, blood, or biopsy material may yield C psittaci. This is not done routinely in clinical laboratories, both because it requires specialized methods and because of the risk to laboratory personnel.

Swabs, scrapings, and tissue specimens should be placed in transport medium. A useful medium has 0.2 mol/L sucrose in 0.02 M phosphate buffer, pH 7.0–7.2, with 5% fetal calf serum. The transport medium should contain antibiotics to suppress bacteria other than Chlamydia species. Gentamicin, 10 μg/mL, vancomycin, 100 μg/mL, and amphotericin B, 4 μg/mL, can be used in combination since they do not inhibit chlamydia. If specimens cannot be processed rapidly, they can be refrigerated for 24 hours; otherwise, they should be frozen at −60°C or colder until processed.

Microscopy and Stains

Cytologic examination is important and useful only in the examination of conjunctival scrapings to diagnose inclusion conjunctivitis and trachoma caused by C trachomatis. Typical intracytoplasmic inclusions can be seen, classically with Giemsa-stained specimens. Fluorescein-conjugated monoclonal antibodies can be used for direct examination of specimens from the genital tract and ocular specimens but are not as sensitive as chlamydial culture or molecular diagnostic tests.

Culture

When culture is required, cell culture techniques are recommended for the isolation of Chlamydia species. Cell culture for C trachomatis and C psittaci usually involves inoculation of the clinical specimens onto cycloheximide-treated McCoy cells, whereas C pneumoniae requires pretreated HL or HEP-2 cells. To detect C trachomatis, immunofluorescence, Giemsa’s stain, or iodine stain is used to search for intracytoplasmic inclusions. Immunofluorescent techniques are the most sensitive of the three stains but require special IF reagents and microscopy. Giemsa is more sensitive than iodine, but the microscopy is more difficult.

Inclusions of C trachomatis stain with iodine, but inclusions of C pneumoniae and C psittaci do not (see Chapter 27). These two species are distinguished from C trachomatis by their different responses to iodine staining and by their susceptibility to sulfonamide. C pneumoniae in culture can be detected by using a genus-specific monoclonal antibody or, even better, a species-specific monoclonal antibody. Serologic techniques for species differentiation are not practical, although C trachomatis can be typed by the microimmunofluorescent method.

Antigen Detection and Nucleic Acid Hybridization

EIAs are no longer recommended for detecting chlamydial antigens in genital tract specimens from patients with sexually transmitted disease. Compared with the more sensitive NAATs for chlamydia (see below), EIAs have much lower sensitivities. Direct fluorescent antibody (DFA) tests may still be useful for testing some specimens from nongenital sites such as conjunctivae in newborns. Nucleic acid amplification tests are available based on PCR, TMA, or strand displacement amplification. These tests are much more sensitive than culture and other nonamplification tests, and have high specificity. (See Chapter 27.)

Serology

The CF test is widely used to diagnose psittacosis. The serologic diagnosis of chlamydial infections is discussed in Chapter 27.

The microimmunofluorescence method is more sensitive than CF for measuring antichlamydial antibodies. The titer of immunoglobulin (Ig) G antibodies can be diagnostic when fourfold titer rises are seen in acute and convalescent sera. However, it may be difficult to show a rise in the IgG titer because of high background titers in the sexually active population. The measurement of IgM antibodies is particularly useful in the diagnosis of C trachomatis pneumonia in neonates. Babies born to mothers with chlamydial infections have serum IgG antichlamydial antibodies from the maternal circulation. Babies with ocular or upper respiratory tract infections have low titers of antichlamydial IgM, whereas babies with chlamydial pneumonia have antichlamydial IgM titers of 1:32 or greater.

Diagnostic virology requires communication between the physician and the laboratory and depends on the quality of specimens and information supplied to the laboratory.

The choice of methods for laboratory confirmation of a viral infection depends on the stage of the illness (Table 47-4). Antibody tests require samples taken at appropriate intervals, and the diagnosis often is not confirmed until convalescence. Virus isolation, antigen detection, or NAAT is required (1) when new epidemics occur, as with influenza; (2) when serologic tests are not useful; and (3) when the same clinical illness may be caused by many different agents. For example, aseptic (nonbacterial) meningitis may be caused by many different viruses; similarly, respiratory disease syndromes may be caused by many viruses as well as by mycoplasmas and other agents.

Diagnostic methods based on NAATs have replaced most but not all virus culture approaches. However, the need for appropriate sample collection and test interpretation will not change. Furthermore, there will be times when recovery of the infectious agent is desired.

Isolation of a virus may not establish the cause of a given disease. Many other factors must be considered. Some viruses persist in human hosts for long periods of time, and therefore, the isolation of herpesviruses, poliovirus, echoviruses, or coxsackieviruses from a patient with an undiagnosed illness does not prove that the virus is the cause of the disease. A consistent clinical and epidemiologic pattern must be established before it can be determined that a particular agent is responsible for a specific clinical picture.

Many viruses are most readily isolated during the first few days of illness. The specimens to be used in virus isolation attempts are listed in Table 47-5. A correlation of virus isolation and antibody presence helps in making the diagnosis, but it is rarely done.

Specimens can be refrigerated for up to 24 hours before virus cultures are done, with the exception of respiratory syncytial and certain other viruses. Otherwise, material should be frozen (preferably at −60°C or colder) if there is a delay in bringing it to the laboratory. Specimens that should not be frozen include (1) whole blood drawn for antibody determination, from which the serum must be separated before freezing; and (2) tissue for organ or cell culture, which should be kept at 4°C and taken to the laboratory promptly.

Virus is present in respiratory illnesses in pharyngeal or nasal secretions. Virus can be demonstrated in the throat fluid and scrapings from the base of vesicular rashes. In eye infections, virus is detectable in conjunctival swabs or scrapings and in tears. Encephalitides are usually diagnosed more readily by serologic means or nucleic acid amplification methods. Arboviruses and herpesviruses are not usually recovered from spinal fluid, but brain tissue from patients with viral encephalitis may yield the causative virus. In illnesses associated with enteroviruses, such as central nervous system disease, acute pericarditis, and myocarditis, viruses can be isolated from feces, throat swabs, or CSF. However, as previously stated, NAATs are the preferred methods for detecting enteroviruses in CSF. DFA tests may be as sensitive as culture for detection of respiratory tract infections with respiratory syncytial virus, influenza viruses A and B, parainfluenza viruses, and adenoviruses. These tests provide answers within a few hours after collection of the specimen compared with days for virus culture. However, these rapid antigen detection methods are being replaced by equally rapid real-time PCR and microarray technologies that allow simultaneous detection of a variety of viruses of interest.

Direct Examination of Clinical Material: Microscopy and Stains

Viral diseases for which direct microscopic examination of imprints or smears has proved useful include rabies and herpes simplex infection and varicella-zoster skin infections. Staining of viral antigens by immunofluorescence in a brain smear and corneal impressions from the rabid animal and from the skin of the nape of the neck of humans is the method of choice for routine diagnosis of rabies.

Virus Culture

A. Preparation of Inocula

Bacteria-free fluid materials such as CSF, whole blood, plasma, or white blood cell buffy coat layer may be inoculated into cell cultures directly or after dilution with buffered phosphate solution (pH 7.6). Inoculation of embryonated eggs or animals for virus isolation is generally performed only in specialized laboratories.

Tissue is washed in media or sterile water, minced into small pieces with scissors, and ground to make a homogeneous paste. Diluent is added in amounts sufficient to make a concentration of 10–20% (weight/volume). This suspension can be centrifuged at low speed to sediment-insoluble cellular debris. The supernatant fluid may be inoculated; if bacteria are present, they are eliminated as discussed below.

Tissues may also be treated with the protease trypsin to release cells from the extracellular matrix, and the resulting cell suspension may be (1) inoculated on an existing tissue culture cell monolayer or (2) cocultivated with another cell suspension of cells known to be virus-free.

If the material to be tested contains bacteria (throat washings, stools, urine, infected tissue, or insects), they must be inactivated or removed before inoculation.

1. Bactericidal agents—Antibiotics are commonly employed in combination with differential centrifugation (see below).

2. Mechanical methods

a. Filters—Millipore-type membrane filters of cellulose acetate or similar inert material are preferred with 20-μm pore size to exclude bacteria.

b. Differential centrifugation—This is a convenient method for removing many bacteria from heavily contaminated preparations of small viruses. Bacteria are sedimented at low speeds that do not sediment the virus, and high-speed centrifugation that sediments the virus. The virus-containing sediment is then resuspended in a small volume.

B. Cultivation in Cell Culture

Cell culture techniques are being replaced by antigen detection methods and NAATs. However, they are still useful and are still practiced in clinical, research, and public health virology laboratories. When viruses multiply in cell culture, they produce biologic effects (eg, cytopathic changes, viral interference, production of a hemagglutinin) that permit identification of the agent.

Test tube cultures are prepared by adding cells suspended in 1–2 mL of nutrient fluid that contains balanced salt solutions and various growth factors (usually serum, glucose, amino acids, and vitamins). Cells of fibroblastic or epithelial nature attach and grow on the wall of the test tube, where they may be examined with the aid of a low-power microscope.

With many viruses, growth of the agent is paralleled by degeneration of these cells (Figure 47-1). Some viruses produce a characteristic cytopathic effect (CPE) in cell culture (shrinking, swelling, rounding of cells, formation of syncytia or clusters), making a rapid presumptive diagnosis possible when the clinical syndrome is known. For example, respiratory syncytial virus characteristically produces multinucleated giant cells (syncytia), whereas adenoviruses produce grapelike clusters of large round cells. Some viruses (eg, rubella virus) produce no direct cytopathic changes but can be detected by their interference with the CPE of a second challenge virus (viral interference). Influenza viruses and some paramyxoviruses may be detected within 24–48 hours if erythrocytes are added to infected cultures. Viruses maturing at the cell membrane produce a hemagglutinin that enables the erythrocytes to adsorb at the cell surface (hemadsorption). The identity of a virus isolate is established with type-specific antiserum, which inhibits virus growth or which reacts with viral antigens.

Some viruses are very slow growers or difficult to culture. Alternative tests instead of culture are used to diagnose such infections (see below).

C. Shell Vial Cultures (Centrifugation-Enhanced Culture)

This method allows for rapid detection of viruses in clinical specimens. It has been adapted for several viruses, including CMV and varicella-zoster virus. For example, CMV can be detected in 18–24 hours, compared with 2–4 weeks for classic cell culture; the sensitivities of shell vial and classic cell cultures for CMV are comparable. Monolayers of the appropriate cell line (eg, MRC-5 cells for CMV) are grown on coverslips in 15 × 45-mm 1-dram shell vials. After inoculation with the specimen, the vials are centrifuged at 700 × g for 40 minutes at room temperature to allow viral attachment to cells. The vials are incubated at 37°C for 16–24 hours, fixed, and reacted with a monoclonal antibody specific for a CMV nuclear protein that is present very early in the culture. Direct or indirect antibody staining methods and fluorescence microscopy are used to examine shell vial cultures. Positive and negative control vials are included in each test run. A modification of the shell vial technique has been developed to allow the simultaneous recovery and detection of multiple respiratory viruses using R-Mix cells. One vial contains (mixes) two cell lines such as human lung carcinoma A549 and mink lung fibroblast Mv1Lu cells. The laboratory will typically inoculate two such vials. After 18–24 hours of incubation, one vial is stained using a “pooled” IF reagent that detects all of the common respiratory viruses. If the stain is positive, then the cells on the coverslip of the second vial are scraped, inoculated onto an eight-well slide, and then stained with individual monoclonal antibody reagents that detect the specific virus. Isolates are not obtained using the shell vial technique. If isolates are needed for susceptibility testing for antiviral drugs, the classic cell culture technique should be used.

Enzyme-Linked Virus-Inducible System (ELVIS)

This test uses a proprietary cell line to detect HSVs in culture. A baby hamster kidney cell line was genetically engineered using the promoter sequence of the HSV UL97 gene and the E coli lacZ gene. When herpes viruses are present in clinical samples, they activate the UL97 promoter, which activates the lacZ gene to produce the enzyme β-galactosidase. When a substrate for the enzyme is added, a blue color is produced indicating the presence of virus. HSV typing can be performed on the positive cultures by adding monoclonal antibodies that detect HSV-1 or HSV-2.

Antigen Detection

The detection of viral antigens is widely used in diagnostic virology. Commercial kits are available to detect many viruses, including herpes simplex I and II, influenza A and B, respiratory syncytial virus, adenoviruses, parainfluenza viruses, rotavirus, and CMV. Multiple types of assays are used as well: EIA, DFA, indirect fluorescent antibody, latex agglutination, etc. The advantages of these procedures are that they allow detection of viruses that do not readily grow in cell culture (eg, rotaviruses, hepatitis A virus) or that grow very slowly (eg, CMV). In general, the antigen detection assays for viruses are less sensitive than the viral culture methods and NAATs. As stated earlier, many of these assays have been or will be replaced by molecular techniques.

Immune Electron Microscopy

Viruses not detectable by conventional techniques may be observed by immune electron microscopy (IEM). Antigen–antibody complexes or aggregates formed between virus particles in suspension are caused by the presence of antibodies in added antiserum and are detected more readily and with greater assurance than individual virus particles. IEM is used to detect viruses that cause enteritis and diarrhea; these viruses generally cannot be cultured by routine virus culture.

Nucleic Acid Amplification and Detection

A wide variety of commercial assays are available to detect viral nucleic acid or to amplify and detect it. These procedures are rapidly becoming the standards for diagnostic virology, supplanting the traditional virus culture and antigen detection techniques. The methods include PCR, reverse transcriptase PCR, and other proprietary methods (see above section on Molecular Diagnostics). The procedures allow detection of viruses (eg, enteroviruses and many others) as well as quantitation of the viruses (eg, HIV-1, CMV, Epstein-Barr virus, hepatitis viruses B and C, and HIV). Data from quantitative assays are used to guide antiviral therapy in multiple viral diseases.

Nucleic Acid Hybridization

Nucleic acid hybridization to detect viruses is highly sensitive and specific. The specimen is transferred to a nitrocellulose membrane, and viral nucleic acid present in the sample is bound; it is then denatured with alkali in situ and hybridized with a labeled viral nucleic acid fragment, and the hybridized products are detected. For rotavirus, which contains double-stranded RNA, the dot hybridization method is even more sensitive than EIA. RNA in heat-denatured fecal samples containing rotavirus is immobilized as above, and in situ hybridization is carried out with labeled single-stranded probes obtained by in vitro transcription of rotavirus.

Nucleic Acid Sequencing

The sequence of viruses can be determined and used to characterize the particular strain infecting a patient. This information is used to predict drug resistance for certain viruses such as HIV, hepatitis C, and CMV. The viral gene sequence is compared to specialized databases containing known resistance mutations, defined through in vitro culture or clinical treatment failures, and a report is generated of the likely resistance pattern for the virus. This testing is useful when the mechanism of drug resistance is known and there are alternate treatment options available for resistant strains.

Measuring the Immune Response to Virus Infection

Typically, a virus infection elicits immune responses directed against one or more viral antigens. Both cellular and humoral immune responses usually develop, and measurement of either may be used to diagnose a viral infection. Cellular immunity may be assessed by dermal hypersensitivity, lymphocyte transformation, and cytotoxicity tests. Humoral immune responses are of major diagnostic importance. Antibodies of the IgM class appear initially and are followed by IgG antibodies. The IgM antibodies disappear in several weeks, whereas the IgG antibodies persist for many years. Establishing the diagnosis of a viral infection is accomplished serologically by demonstrating a rise in antibody titer to the virus or by demonstrating antiviral antibodies of the IgM class (see Chapter 8). The methods used include the neutralization (Nt) test, the CF test, the hemagglutination inhibition (HI) test, and the IF test, passive hemagglutination, and immunodiffusion.

Measurement of antibodies by different methods does not necessarily give parallel results. Antibodies detected by the CF test are present during an enterovirus infection and in the convalescent period, but they do not persist. Antibodies detected by the Nt test also appear during infection and persist for many years. Assessment of antibodies by several methods in individuals or groups of individuals provides diagnostic information as well as information about epidemiologic features of the disease.

Serologic tests for viral diagnosis are most useful when the virus has a long incubation period prior to the appearance of clinical manifestations. A partial list of such viruses includes Epstein-Barr virus, the hepatitis viruses, and HIV. Typically, testing for antibodies to these viruses is the first step in diagnosis and may be followed later, in most cases, by nucleic acid amplification that is used to assess the levels of circulating virus as an estimate of the level of infection and/or response to specific antiviral therapies. Another important utility of serologic tests is to assess one’s vulnerability or prior exposure to a virus and the potential for reactivation in the context of immunosuppression or organ transplantation.

Algorithms for diagnosis of viral infections have changed as newer assays with improved performance have become available. A good example of this is the evolution of HIV diagnostic testing. HIV immunoassays have progressed from first-generation to third-generation assays which have increased sensitivity and specificity. Fourth-generation assays added the detection of HIV p24 antigen to allow for earlier identification of infections. Before 2014, diagnosis of HIV required confirmation of positive serology results by Western blot, which involves binding of patient serum antibodies to HIV proteins separated by protein electrophoresis. Specific patterns of antibody binding determine a positive, negative, or indeterminate Western blot. The new algorithm uses a combined HIV-1/2 antigen/antibody immunoassay screening test followed by specific HIV-1/HIV-2 differentiation assay. Samples that are negative or indeterminate by the differentiation assays are then subjected to nucleic acid testing to detect acute infections in the window period, where viral nucleic acid is present but antibodies have not yet formed. The current HIV testing algorithm is highly sensitive and specific, and can identify early infections prior to the development of patient antibodies. Universal screening for HIV has been recommended in adults and adolescents, with repeat testing of pregnant women and high-risk patients, in order to reduce overall HIV transmission.