Microbiologic Studies

OVERVIEW OF MICROBIOLOGIC STUDIES

Diagnostic Testing and Microbes

Microorganisms that “cause infectious disease” are defined as pathogens. Organisms that are pathogenic under one set of conditions may, under other conditions, reside within or on the surface of the body without causing disease. When organisms are present but do not cause harm to the host, they are considered commensals. When organisms multiply and cause tissue damage, they are considered pathogens, with the potential for causing or increasing a pathogenic process (Table 7.1). Many newly discovered organisms are clinically relevant. Some of these organisms, formerly considered insignificant contaminants or commensals, have taken on roles as causative agents for opportunistic diseases in patients with human immunodeficiency virus (HIV) infection or other immunodeficiency syndromes or diseases associated with a compromised health state. Consequently, virtually any organism recovered in pure culture from a body site must be considered a potential pathogen.

Basic Concepts of Infectious Disease

Infectious processes demonstrate observable physiologic responses to the invasion and multiplication of the offending microorganisms. Once an infectious disease is suspected, appropriate cultures should be done or nonculture techniques should be used, such as serologic testing for antigens and antibodies, monoclonal antibodies, and DNA probes. Proper specimen collection and appropriate blood and skin tests are necessary to detect and diagnose the presence of the microorganism.

Opportunity for infection depends on host resistance, organism volumes, and the ability of the organism to find a portal of entry and to overcome host defenses, invade tissues, and produce toxins. Organisms may become seated in susceptible persons through inhalation, ingestion, direct contact, inoculation, breaks in natural skin or mucous membrane barriers, changes in organism volumes, alterations in normal flora balances, or changes in other host defense mechanisms.

Host Factors

The development of an infectious disease is influenced by the patient’s general health, normal defense mechanisms, previous contact with the offending organism, past clinical history, and type and location of infected tissue. Mechanisms of host resistance are detailed in the following lists:

Primary host defenses
- Anatomic barriers
  - Intact skin surfaces
  - Nose hairs
  - Respiratory tract cilia
  - Coughing and flow of respiratory tract fluids and mucus
  - Swallowing and gastrointestinal (GI) tract peristalsis
- Physiologic barriers
  - High or low pH and oxygen tension (prevents proliferation of organisms)
  - Chemical inhibitors to bacterial growth (e.g., proteases)
  - Bile acids
  - Active lysozymes in saliva and tears
  - Fatty acids on skin surfaces

Secondary host defenses (physiologic barriers)

Responses of complement, lysozymes, opsonins, and secretions
Phagocytosis
Immunoglobulin A (IgA), IgG, and IgM antibody formation
Cell-mediated immune responses

TABLE 7.1 Some Common Pathogens Detectable in Body Tissues and Fluids by Diagnostic Methods

Nasopharynx and Oropharynx	Sputum	Feces
β-Hemolytic streptococci Bordetella pertussis Mycoplasma spp. Moraxella catarrhalis Herpes simplex virus Pseudomonas spp. Candida albicans Corynebacterium diphtheriae Haemophilus influenzae Neisseria meningitidis Streptococcus pneumoniae Staphylococcus aureus Enterobacteriaceae Cryptococcus neoformans Respiratory syncytial virus Influenza viruses Parainfluenza viruses Adenovirus Rhinovirus Coronavirus	Blastomyces dermatitidis Bordetella pertussis Candida albicans Coccidioides immitis Influenza viruses Streptococcus pneumoniae Pseudomonas spp. Haemophilus influenzae β-Hemolytic streptococci Histoplasma capsulatum Klebsiella spp. Mycobacterium spp. Yersinia pestis Francisella tularensis Staphylococcus aureus Mycoplasma spp. Legionella spp. Chlamydophila pneumoniae Pneumocystis	Campylobacter jejuni Clostridium botulinum Entamoeba histolytica Escherichia coli Salmonella spp. Shigella spp. Staphylococcus aureus Vibrio cholerae Vibrio comma Vibrio parahaemolyticus Yersinia enterocolitica Clostridium difficile Rotavirus Hepatitis A, B, and C Giardia lamblia Cryptosporidium spp. Norovirus Aeromonas sp. Plesiomonas sp. Leptospira
Urine
Streptococcus agalactiae Escherichia coli, other Enterobacteriaceae Enterococcus spp. Neisseria gonorrhoeae Mycobacterium tuberculosis	Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus saprophyticus Salmonella and Shigella spp. Trichomonas vaginalis Candida albicans and other yeasts Staphylococcus epidermidis
Skin	Ear
Bacteroides spp. Clostridium spp. Fungi Pseudomonas spp. Staphylococcus aureus Streptococcus pyogenes Varicella zoster virus Sarcoptes scabiei Herpes simplex virus Bacillus anthracis Treponema pallidum	Aspergillus fumigatus Candida albicans and other yeast Enterobacteriaceae β-Hemolytic streptococci Streptococcus pneumoniae Pseudomonas aeruginosa Staphylococcus aureus Moraxella catarrhalis Mycoplasma pneumoniae Peptostreptococcus spp. Bacteroides fragilis Fusobacterium nucleatum Influenza virus Respiratory syncytial virus (RSV)
Cerebrospinal Fluid	Vaginal Discharge	Urethral Discharge
Bacteroides spp. Cryptococcus neoformans Haemophilus influenzae Mycobacterium tuberculosis Neisseria meningitidis Streptococcus pneumoniae Enteroviruses Listeria monocytogenes Streptococcus agalactiae (Group B) Staphylococcus spp. Escherichia coli Herpes simplex virus Mycoplasma	β-Hemolytic streptococci Candida albicans Gardnerella vaginalis Listeria monocytogenes Mycoplasma spp. Human papilloma virus Neisseria gonorrhoeae Treponema pallidum Herpes simplex virus Trichomonas vaginalis Chlamydia trachomatis	Chlamydia trachomatis Coliform bacilli Herpes simplex virus Neisseria gonorrhoeae Treponema pallidum Trichomonas vaginalis Mycoplasma spp. Ureaplasma urealyticum Human papillomavirus Mobiluncus spp. and other anaerobes

Factors decreasing host resistance
- Age: The very young and the very old are more susceptible.
- Presence of chronic disease (e.g., cancer, cardiovascular disease, diabetes)
- Use or history of certain therapeutic modalities, such as radiation, chemotherapy, corticosteroids, antibiotics, or immunosuppressants
- Toxins, including alcohol, street drugs, legitimate therapeutic drugs, venom or toxic secretions from a reptile or insect, or other nonhuman bites or punctures
- Others, including excessive physical or emotional stress states, nutritional state, and presence of foreign material at the site

COLLECTION AND TRANSPORT OF SPECIMENS

General Principles

The health care professional is responsible for collecting specimens for diagnostic examinations. Because procedures vary, check institutional protocols for specimen retrieval, transport, preservation, and reporting of test results.

Specimens for bacterial culture should be representative of the disease process. Also, sufficient material must be collected to ensure an accurate examination. As an example, serous drainage from a diabetic foot ulcer with possible osteomyelitis may yield inaccurate results. In this case, a bone biopsy or purulent drainage of infected tissue would be a better specimen. Likewise, if there is a lesion of the skin and subcutaneous tissue, material from the margin of the lesion rather than the central part of the lesion would be more desirable. If a purulent sputum sample cannot be obtained to aid in the diagnosis of pneumonia, blood cultures, pleural fluid examination, and bronchoalveolar lavage (BAL) specimens are also acceptable.

It is imperative that material be collected where the suspected organism is most likely to be found, with as little contamination from normal flora as possible. For this reason, certain precautions must be followed routinely:

Observe standard precautions. Clean the skin starting centrally and going out in larger circles. Repeat several times, using a clean swab or wipe each time. If 70% alcohol is used, it should be applied for 2 minutes. Tincture of iodine requires only 1 minute of cleansing.
Bypass areas of normal flora; culture only for a specific pathogen.
Collect fluids, tissues, skin scrapings, and urine in sterile containers with tight-fitting lids. Polyester-tipped swabs in a collection system containing an ampule of Stuart’s transport medium ensure adequacy of the specimen for 72 hours at room temperature.
Place the specimen in a biohazard bag.

Sources of Specimens

Microbiologic specimens may be collected from many sources, such as blood, tissue, pus or wound exudates or drainage, urine, sputum, feces, genital discharges or secretions, cerebrospinal fluid (CSF), and eye or ear drainage. During specimen collection, these general procedures should be followed:

Label specimens properly with the following information (institutional requirements may vary):
- Patient’s name, age, sex, address, hospital identification number, and physician’s full name
- Specimen source (e.g., throat, conjunctiva)
- Time of collection
- Specific studies ordered
- Clinical diagnosis; suspected microorganisms
- Patient’s history
- Patient’s immune state
- Previous and current infections
- Previous or current antibiotic therapy
- Isolation status—state type of isolation (e.g., contact, respiratory, wound)
- Other requested information pertinent to testing
2. Avoid contaminating the specimen; maintain aseptic or sterile technique as required:
- Special supplies may be required:
  - For anaerobes, sterile syringe aspiration of pus or other body fluid
  - Anaerobic transport containers for tissue specimens
- Sterile specimen containers
- Precautions to take during specimen collection include:
  - Care to maintain cleanliness outside container surfaces
  - Use of appropriately fitting covers or plugs for specimen tubes and bottles
  - Replacement of sterile plugs and caps that have become contaminated
  - Observation of standard precautions
- Ensure the preservation of specimens by delivering them promptly to the laboratory. Many specimens may be refrigerated (not frozen) for a few hours without any adverse effects. Note the following exceptions:
  - Urine culture samples must be refrigerated.
  - CSF specimens should be transported to the laboratory as soon as possible. If this is problematic, the culture should be incubated (meningococci do not withstand refrigeration). Both culture bottles must be maintained at room temperature prior to being placed in the analyzer.
  - Blood culture bottles must be maintained at room temperature.
- Transport specimens quickly to the laboratory to prevent desiccation of the specimen and death of the microorganisms.
  - For anaerobic cultures, no more than 10 minutes should elapse between time of collection and culture. Anaerobic specimens should be placed into an anaerobic transport container.
  - Feces suspected of harboring Salmonella or Shigella organisms should be placed in a special transport medium, such as Cary-Blair, if culturing of the specimen will be delayed longer than 30 minutes.
- Ensure that specimen quantity is adequate. With few exceptions, the quantity of the specimen should be as large as possible. When only a small quantity is available, swabs should be moistened with sterile saline just before collection, especially for nasopharyngeal cultures.
- Handle specimen collection in the following way:
  - Submit entire fluid specimen collected. Do not submit fluids on swabs.
  - Whenever possible, specimens should be collected before antibiotic regimens are instituted; for example, complete all blood culture sampling before starting antibiotic therapy.

Transport of Specimens by Mail

Several kits containing transport media are available for use when there is a significant delay between collection and culturing. Culture swabs (containing transport medium) are available for bacterial, viral, and anaerobic collection of specimens. Some laboratories provide Cary-Blair and polyvinyl alcohol (PVA) or non-mercury-based fixative transport vials for stool collection for culture and ova and parasite examination, respectively. Depending on the request, some specimens may have to be shipped in a Styrofoam box with refrigerant packs. This is especially true for specimens to be tested for viral examination. It is prudent to consult the reference laboratory to which specimens will be sent for information on proper collection and shipment.

According to the Code of Federal Regulations (49 CFR), a viable organism or its toxin or a diagnostic specimen (volume <50 mL) must be placed in a secure, closed, watertight container that is then enclosed in a second secure, watertight container. Biohazard labels should be placed on the outside of the container.

Specimens that are to be transported within an institution should be placed in a sealed biohazard bag. Ideally, the requisition should accompany the specimen but not be sealed inside the bag.

Diagnosis of Bacterial Disease

Bacteriologic studies attempt to identify the specific organism causing an infection (Table 7.2). This organism may be specific to one disease, such as Mycobacterium tuberculosis for tuberculosis (TB), or it may cause a variety of infections, such as those associated with Staphylococcus aureus. Antibiotic susceptibility studies then determine the responses of the specific organism to various classes and types of antibiotics. An antibiotic that inhibits bacterial growth is the logical choice for treating the infection.

Some questions that need to be asked when searching for bacteria as the cause of a disease process include the following: (1) Are bacteria responsible for this disease? (2) Is antimicrobial therapy indicated? Most bacteria-related diseases have a febrile course. From a practical standpoint during evaluation of the febrile patient, the sooner a diagnosis can be reached and the sooner a decision can be made concerning antimicrobial therapy, the less protracted the period of recovery.

Anaerobic bacterial infections are commonly associated with localized necrotic abscesses: they may yield several different strains of bacteria. Because of this, the term polymicrobic disease is sometimes used to refer to anaerobic bacterial diseases. This view is in sharp contrast to the “one organism, one disease” concept that characterizes other infections, such as typhoid fever, cholera, or diphtheria. Isolation and identification of the different strains of anaerobic bacteria and susceptibility studies may be desirable so that appropriate therapy may be given.

TABLE 7.2 Bacterial Diseases and Their Laboratory Diagnosis

Disease	Causative Organism	Source of Specimen	Diagnostic Tests
Anthrax Brucellosis (undulant fever)	Bacillus anthracis Brucella melitensis, Brucella abortus, Brucella suis	Blood, sputum, skin Blood, bone marrow, CSF, tissue, lymph node, urine	Gram stained, smear and culture Culture, serology
Bubonic plague	Yersinia pestis	Bubos (enlarged and inflamed lymph nodes), blood, sputum	Skin, blood, and sputum smear; culture
Chancre	Haemophilus ducreyi	Genital lesion biopsy	Lesion smear and culture; biopsy
Cholera	Vibrio cholerae	Feces	Stool smear and culture
Psittacosis	Chlamydia psittaci	Blood, sputum, lung tissue	Culture, smear, serologic tests
Diphtheria	Corynebacterium diphtheriae	Nasopharynx	Nasopharyngeal smear and culture
Erysipeloid	Erysipelothrix rhusiopathiae	Lesion, blood	Smear and culture
Gonorrhea	Neisseria gonorrhoeae	Cervix, urethra, CSF, blood, joint fluid, throat	Smear and culture, nucleic acid amplification assay
Granuloma inguinale (donovanosis)	Calymmatobacterium granulomatis	Groin lesion	Smear
Gastritis, gastric ulcer	Helicobacter pylori	Gastric tissue biopsy	Culture, biopsy, with histopathologic exam
Relapsing fever	Borrelia recurrentis	Peripheral blood	Direct examination, serology
Lyme disease	Borrelia burgdorferi	Blood	Serologic test
Legionnaires’ disease	Legionella pneumophila	Sputum	Culture, direct fluorescent antibody; serologic test, urine antigen test, nucleic acid amplification assay
Leprosy (Hansen’s disease)	Mycobacterium leprae	Skin scrapings	Acid fast smear, biopsy with histopathologic exam
Lymphogranuloma venereum	Chlamydia trachomatis	Genitalia, conjunctiva, urethra, urine	Culture, DNA probe, amplified test
Listeriosis	Listeria monocytogenes	Blood, CSF, amniotic fluid, placenta, vagina	Smears and culture, serologic test
Pneumonia	Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae	Bronchoscopy, secretions, sputum, blood, lung aspirate or biopsy, pleural fluid	Smear and culture
Strep throat, scarlet fever, impetigo	Streptococcus pyogenes	Throat, lesion	Culture, serologic test
Tetanus	Clostridium tetani	Wound	Smear and culture
Toxic shock syndrome	Staphylococcus aureus	Tissue	Culture
Tuberculosis	Mycobacterium tuberculosis	Sputum, gastric washings, urine, CSF	Smear and culture of sputum, gastric washings, urine, and CSF; skin test, QuantiFERON-TB assay
Tularemia	Francisella tularensis	Skin, lymph node, ulcer tissue biopsy, sputum, bone marrow	Serologic test, smear and culture
Typhoid	Salmonella typhi	Blood (after first week of infection); feces (after second week of infection)	Culture and serologic test
Whooping cough	Bordetella pertussis	Nasopharyngeal swab	Culture, fluorescent antibody test, nucleic acid amplification assay, serologic test
Nocardiosis	Nocardia asteroides	Sputum, lesion	Smear and culture
Mycoplasma	Mycoplasma pneumoniae	Sputum, nasopharyngeal and throat swabs	Serology, culture, PCR

Studies of the Susceptibility of Bacteria to Antimicrobial Agents

The susceptibility test detects the type and amount of antibiotic or chemotherapeutic agent required to inhibit bacterial growth. Often, culture and susceptibility tests are ordered together. Susceptibility studies also may be indicated when an established regimen or treatment is to be altered.

A common and useful test for evaluating antibiotic susceptibility is the disk diffusion method. A set of antibiotic-impregnated disks on agar is inoculated with a culture derived from the specific bacteria being tested. After a suitable period of incubation, the degree of bacterial growth within the different antibiotic zones on the disks is determined and measured. Growth zone diameters, measured in millimeters, are correlated to the minimum inhibitory concentration (MIC) to determine whether the organism is truly susceptible to the antibiotic. Another method is a broth dilution test. The organism is grown in the presence of doubling dilutions of the antibiotic. The lowest concentration of the antibiotic that inhibits the organism’s growth is the MIC. Many commercial systems are based on this method.

Clinical Implications

The terms sensitive and susceptible imply that an infection caused by the bacterial strain tested is likely to respond favorably in the presence of the indicated antimicrobial agent.
The terms intermediate, partially resistant, and moderately susceptible mean that the bacterial strain tested is not completely inhibited by therapeutic concentrations of a test drug.
Indeterminate means that the bacterium has an MIC that approaches achievable blood and tissue concentrations. It implies clinical efficacy in body sites where the antibiotic is physiologically concentrated. The intermediate category also includes a buffer zone, which should prevent major errors due to technical factors.
The term resistant implies that the organism is not inhibited by the antibiotic. This infection is not likely to respond to treatment.
Some antimicrobial agents act in a bactericidal manner, meaning that they kill the organism. Others act in a bacteriostatic manner, meaning that they inhibit growth of the organism but do not necessarily kill it.
- Bactericidal agents
  - Aminoglycosides
  - Cephalosporins
  - Metronidazole
  - Penicillins
  - Quinolones
  - Rifampin
  - Vancomycin
- Bacteriostatic agents
  - Chloramphenicol
  - Erythromycin
  - Sulfonamides
  - Tetracycline
- Emergence of strains of penicillin-resistant Neisseria gonorrhoeae, carbapenem-resistant enterobacteriaceae (CRE) and extended spectrum beta lactamases (ESBLs), methicillin-resistant (or oxacillin-resistant) S. aureus, amikacin-resistant Pseudomonas spp. or other gram-negative rods, and vancomycin-resistant Enterococcus spp. presents challenges to the clinician in regard to treatment. Many hospitals screen for methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) spp. so as to isolate patients infected with these organisms.

Diagnosis of Mycobacterial Infections

The genus Mycobacterium contains several species of bacteria that are pathogenic to humans (Table 7.3). For example, M. tuberculosis is spread from person to person through inhalation of airborne respiratory secretions containing mycobacteria expelled during coughing, sneezing, or talking. In patients with the acquired immunodeficiency syndrome (AIDS), Mycobacterium avium-intracellulare (MAI) complex is acquired through the GI tract, often through ingestion of contaminated water or food.

The disease progression of mycobacteriosis, particularly in patients with AIDS, is rapid (a few weeks). This short time span has required new methods for rapid recovery and identification of mycobacteria so that antibiotic therapy can be instituted promptly. These newer techniques involve the use of instruments that shorten the growth period for mycobacteria to 1 to 2 weeks. Isotopic nucleic acid probes are available for culture identification of M. tuberculosis, MAI complex, Mycobacterium kansasii, and Mycobacterium gordonae. Nucleic acid amplification assays, which use DNA technology to detect mycobacteria directly in clinical specimens, are also available to clinical laboratories.

A disturbing problem that has arisen since the resurgence of TB among persons with AIDS is the appearance of multidrug-resistant M. tuberculosis strains.

Reference Values

Normal

Negative results on acid-fast bacillus (AFB) smear or culture

Collection of Specimens

Sputum and bronchial aspirates and lavages are the best samples for diagnosis of pulmonary infection. Purulent sputum (5 to 10 mL) from the first productive cough of the morning should be expectorated into a sterile container. If the specimen is not processed immediately, it should be refrigerated. Pooled specimens collected over several hours are not acceptable. For best results, three specimens should be collected over several days. A prerequisite of good specimen collection is the use of sterile, sturdy, leak-proof containers placed into biohazard bags.

TABLE 7.3 Mycobacterial Infections and Their Laboratory Diagnosis

Causative Organism	Source of Specimen	Diagnostic Test
Mycobacterium tuberculosis	Sputum, urine, CSF, tissue, bone marrow	Culture and smear; skin test; DNA probe; nucleic acid amplification assay
Mycobacterium avium-intracellulare	Sputum, stool, CSF, tissue, blood, semen, lymph nodes	Culture and smear; DNA probe
Mycobacterium kansasii	Skin, joint, lymph nodes, sputum, tissue	Culture and smear
Mycobacterium leprae	CSF, skin, bone marrow, lymph nodes	Histopathologic examination of lesion
Mycobacterium marinum	Joint lesion, skin	Culture and smear
Mycobacterium xenopi	Sputum	Culture and smear
Mycobacterium fortuitum	Surgical wound, bone, joint, tissue, sputum	Culture and smear
Mycobacterium chelonae	Surgical wound, sputum, tissue	Culture and smear

AFB smears and cultures are done to determine whether TB-like symptoms are due to M. tuberculosis infection or infection from another mycobacterium and to aid in determining whether the TB is intrapulmonary or extrapulmonary.
If the patient is unable to produce sputum, an early-morning gastric sample may be aspirated and cultured. This specimen must be hand-delivered to the laboratory to be processed or neutralized immediately.
Patients with suspected renal disease should provide early-morning urine specimens collected for 3 days in a row. Pooled 24-hour urine collections are not recommended. Unless processed immediately, the specimen should be refrigerated.
If TB meningitis is suspected, at least 10 mL (2 mL in children) of CSF should be obtained.
Sterile body fluids, tissue biopsy samples, and material aspirated from skin lesions are acceptable specimens for mycobacterial cultures. Tissue should be placed in a neutral transport medium to avoid desiccation. Swab specimens are not suitable for mycobacterial culture.
Feces are commonly the first specimens from which MAI complex can be isolated in a patient with disseminated disease. An acid-fast stain can be performed directly.
MAI complex organisms can also be isolated from the blood of immunosuppressed patients.

Diagnosis of Mycobacterium tuberculosis

These tests diagnose TB-infected persons who are at increased risk for TB development and who will benefit from treatment. The QuantiFERON-TB test (QFT-G) is a new test used to detect latent TB. The QFT-G measures interferon-γ (IFN-γ), a component of cell-mediated immune activity to TB response. Tuberculin skin testing (TST) screens for new or latent TB infection in high-risk groups. The TST measures lymphocyte response to TB in persons sensitized to the TB antigen. This test has been used for years and measures a delayed hypersensitivity response (48 to 72 hours).

Reference Values

Normal

QuantiFERON-TB Gold Test (QFT-G):

Negative: <0.35 IU/mL IFN-γ

Positive: ≥0.35 IU/mL IFN-γ

Tuberculin skin testing (TST): negative for cutaneous hypersensitivity—area of induration, <10 mm

Clinical Implication

A positive QFT-G result indicates that TB infection is likely.
A negative QFT-G result indicates that TB infection is unlikely but cannot be excluded.

Interfering Factors

Diabetes, silicosis, and chronic renal failure may decrease responsiveness to both the TST and QFT-G tests.
Treatment with immunosuppressive drugs has been shown to decrease the response to the TST.
Some of the hematologic disorders, such as leukemia and lymphoma, may decrease response to the TST and QFT-G test.

Diagnosis of Rickettsial Disease

Rickettsiae are small, gram-negative coccobacilli that structurally resemble bacteria but are one tenth to one half as large. Polychromatic stains (Giemsa stain) are better than simple stains or the Gram stain for demonstrating rickettsiae in cells.

Rickettsiosis is the general name given to any disease caused by rickettsiae (Table 7.4). These organisms are considered to be obligate intracellular parasites; that is, they cannot exist anywhere except inside the bodies of living organisms. Diseases caused by rickettsiae are transmitted by arthropod vectors, such as lice, fleas, ticks, or mites. Rickettsial diseases are divided into the following general groups:

Typhus-like fevers
Spotted fever
Scrub typhus
Q fever
Other rickettsial diseases

Q fever, caused by Coxiella burnetii, is characterized by an acute febrile illness, severe headache, rigors, and possibly pneumonia or hepatitis. It can cause encephalitis in children and has been isolated in breast milk and in the placenta of infected mothers, making it possible for a fetus to be infected in utero. Both complement fixation and fluorescent antibody tests can detect antibodies to the organism. C. burnetii displays an antigenic variation during an infection. Phase I antibodies are preponderant during the chronic phase, whereas phase II antibodies predominate during the acute phase. A diagnosis is made when the phase I titer in a convalescent serum specimen is four times greater than that in an acute serum specimen.

Early diagnosis of rickettsial infection is usually based on observation of clinical symptoms such as fever, rash, and exposure to ticks. Biopsy specimens of skin tissue from a patient with suspected Rocky Mountain spotted fever can be tested with an immunofluorescent stain and diagnosed 3 to 4 days after symptoms appear. Signs and symptoms include the following:

Fever
Skin rashes
Parasitism of blood vessels
Prostration
Stupor and coma
Headache
Ringing in the ears
Dizziness

TABLE 7.4 Rickettsial Diseases and Their Laboratory Diagnosis

Disease		Geographic Distribution	Natural Cycle		Transmission to Humans	Serologic Diagnosis
Group/Type	Agent	Geographic Distribution	Arthropod	Mammal	Transmission to Humans	Serologic Diagnosis
Epidemic typhus^*	Rickettsia prowazekii	Worldwide	Body louse	Human	Infected louse feces into broken skin	Positive group- and typespecific serologic test
Endemic (murine) typhus	Rickettsia typhi	Worldwide	Flea	Rodents	As above	Positive group- and typespecific serologic test
Spotted fever, Rocky Mountain spotted fever	Rickettsia rickettsii	Western hemisphere	Ticks	Wild rodents, dogs	Tick bite	Serologic tests—IFA or latex agglutination
North Asian tickborne rickettsiosis	Rickettsia sibirica	Siberia, Mongolia	Ticks	Wild rodents	Tick bite	Complement fixation
Boutonneuse fever	Rickettsia conorii	Africa, Europe, Mideast, India	Ticks	Wild rodents	Tick bite	Positive group- and typespecific serologic test
Queensland tick typhus	Rickettsia australis	Australia	Ticks	Marsupials, wild rodents	Tick bite	Complement fixation
Rickettsial pox	Rickettsia akari	North America, Europe	Bloodsucking	Mouse, other rodents	Mite bite	Microimmunofluorescence
Scrub typhus	Rickettsia tsutsugamushi	Asia, Australia, Pacific Islands	Trombiculid	Wild rodents	Mite bite	Specific complement fixation positive in about 50% of patients, and indirect immunofluorescence
Ehrlichiosis	Ehrlichia canis, Ehrlichia sennetsu	Southeast Asia	Ticks	Human	Tick bite	PCR amplification, peripheral blood smears
Q fever	Coxiella burnetii	Worldwide	Ticks	Small mammals, cattle, sheep, and goats	Inhalation of dried, infected material, milk, products of conception	Positive for complement fixation phases I and II
Trench fever	Rochalimaea quintana	Europe, Africa, North America	Body louse	Human	Infected louse feces into broken skin	PCR, culture and serologic tests
Oroya fever	Bartonella bacilliformis	Peru, Ecuador, Columbia, Brazil	Sand fly	Human	Bite of sand fly	PCR, culture, serologic tests
^*Recurrence years after original attack of epidemic typhus.

NOTE

Rickettsial diseases are often characterized by an incubation period of 10 to 14 days, followed by an abrupt onset of the signs and symptoms listed, in a patient with a history of arthropod bites. Cultures of rickettsia are performed only in reference laboratories. Rickettsial infections usually are diagnosed by serologic methods, using acute and convalescent serum specimens. A fourfold rise in serum antibody titer is preferable, but a single titer greater than 1:64 is highly suggestive of infection (see Chapter 8).

Diagnosis of Parasitic Disease

About 70 species of animal parasites commonly infect the human body (Table 7.5). More than half of these can be detected by examination of stool specimens because the parasites inhabit the GI tract and its environs. Of the parasites that can be diagnosed by stool examinations, about one third are single-celled protozoa, and two thirds are multicellular worms. Only six or seven types of intestinal protozoa are clinically important, but almost all of the worm classes are potentially pathogenic.

Diagnosis of parasites begins with ova and parasite examination. Other diagnostic options include sigmoidoscopy smears, biopsies, barium radiologic studies, and serologic tests. Collection of fecal specimens for parasites should be done before administration of barium sulfate, mineral oil, bismuth, antimalarial drugs, and some antibiotics (e.g., tetracycline). For ova and parasite examination, ideally, one specimen should be collected every other day for a total of three specimens. At the most, these specimens should be gathered within 10 days.

For detection of Giardia, other diagnostic tests such as the Entero-Test capsule (string test) and duodenal aspiration or biopsy may be necessary. The Entero-Test consists of a gelatin capsule containing a coiled length of nylon yarn. The capsule is swallowed, the gelatin dissolves, and the weighted string is carried into the duodenum. After about 4 hours, the string is withdrawn, and the accompanying mucus is examined microscopically for Giardia. Duodenal fluid also can be submitted by the physician to be examined for Giardia and Strongyloides stercoralis. The specimen should contain no preservatives and should be examined for organisms within 1 hour after collection.

Cryptosporidium parvum has long been recognized as an animal parasite but is also capable of infecting humans, especially physically compromised patients. Organisms have been recovered from the gallbladder, lungs, and stool.

Collection of Specimens

Multiple specimens may be necessary to detect a parasitic infection.
Most parasites found in humans are identified in blood or feces but may also be evident in urine, sputum, tissue fluids, or biopsy tissues.

Fecal specimens should not be contaminated with water or urine. All specimens should be labeled with the patient’s name, clinician’s name, identification number (if applicable), and date and time collected. Various commercial collection systems are available to allow collection of specimens at home, in a nursing institution, or in a hospital setting. Clear instructions should be communicated and given in writing to the patient to ensure proper collection. See Chapter 4, Stool Studies, for more information.

TABLE 7.5 Parasitic Diseases and Their Laboratory Diagnosis

Disease	Causative Organism	Source of Specimen	Diagnostic Tests
Amebiasis	Entamoeba histolytica	Stool, liver	Stool smear, rectal biopsy, serologic test, antigen test
Ascariasis	Ascaris lumbricoides	Stool, sputum	Ova and parasite examination, antigen test, rectal biopsy, serologic test
Cestodiasis of intestine (tapeworm disease)	Taenia saginatus, Taenia solium, Diphyllobothrium, Hymenolepis nana, Hymenolepis diminuta	Stool	Ova and parasite examination, Scotch tape test for Enterobius vermicularis
Chagas’ disease	Trypanosoma cruzi	Blood, spinal fluid	Giemsa- or Wright-Giemsa-stained smear
Cryptosporidiosis	Cryptosporidium parvum	Stool, lung, gallbladder	Ova and parasite examination, antigen test, direct fluorescent antibody test
Cysticercosis	Taenia solium larvae	Muscle and brain	Muscle and brain cyst biopsy, serology
Echinococcosis	Echinococcus granulosus	Sputum and urine, liver, spleen	Ova and parasite examination, direct microscopic examination, serologic test, Casoni’s skin test; liver and bone biopsy
Enterobiasis (pinworm disease)	Enterobius vermicularis	Stool	Scotch tape test
Filariasis	Wuchereria bancrofti, Brugia malayi, Loa loa	Blood	Blood smear, lymph node biopsy, serologic test
Giardiasis	Giardia lamblia	Stool, duodenal aspirate or biopsy	Ova and parasite examination, antigen test, direct fluorescent antibody test, microscopic examination of Entero test
Hookworm	Ancylostoma duodenale, Necator americanus	Stool	Ova and parasite examination
Isosporiasis Kala-azar	Isospora belli Leishmania donovani	Stool Liver, bone marrow, blood	Ova and parasite examination Giemsa- or Wright-Giemsa-stained smear and culture, lymph node and spleen biopsy
Malaria	Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale	Blood, bone marrow	Giemsa- or Wright-Giemsa-stained smear
Acanthamoebiasis	Acanthamoeba culbertsoni	CSF, corneal biopsy or scraping	Smear and tissue culture
Naegleriasis	Naegleria fowleri	CSF	Smear
Sarcocystis	Sarcocystis hominis or Sarcocystis suihominis	Stool	Ova and parasite examination
Blastocystis	Blastocystis hominis	Stool	Ova and parasite examination
Onchocerciasis	Onchocerca volvulus	Skin	Skin biopsy with histopathologic exam
Paragonimiasis	Paragonimus westermani	Sputum, stool	Ova and parasite examination, serologic test, skin test
Scabies	Sarcoptes scabiei	Skin	Skin smear, direct examination
Schistosomiasis of intestine and bladder	Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium	Stool, urine	Ova and parasite examination, serologic test, skin test, rectal, bladder, and liver biopsy
Strongyloidiasis	Strongyloides stercoralis	Stool, duodenal aspirate	Ova and parasite examination, serologic test
Toxoplasmosis	Toxoplasma gondii	Blood, tissue, CSF	Serologic test, tissue smear, biopsy
Trichinosis	Trichinella spiralis	Muscle	Serologic test, skin test, muscle biopsy
Trichomoniasis	Trichomonas vaginalis	Vagina, bladder, urethra	Vaginal and urethral smear and culture, DNA probe
Trichuriasis	Trichuris trichiura	Stool	Ova and parasite examination
Trypanosomiasis	Trypanosoma rhodesiense, Trypanosoma gambiense	Blood, spinal fluid, lymph node	Blood, spinal fluid and lymph node smear, serologic test
Visceral larva migrans	Toxocara canis, Toxocara cati	Liver	Serologic test, skin test, liver biopsy
Trematodes	Fasciola hepatica, Clonorchis sinensis, Fasciolopsis buski	Stool	Ova and parasite examination

When sputum is collected for ova and parasites, it should be “deep sputum” from the lower respiratory tract. It should be collected early in the morning, before the patient eats or brushes the teeth, and immediately delivered to the laboratory. See Appendix B, Guidelines for Specimen Transport and Storage, for more information.

Clinical Considerations

General considerations
- Eosinophilia may be an indicator of parasitic infection.
- Protozoa and helminths, particularly larvae, may be found in organs, tissues, and blood.
Specimen-related considerations
- Hepatic puncture can reveal visceral leishmaniasis. Liver biopsy may yield toxocaral larvae and schistosomal worms and eggs. Hepatic abscess material from the peripheral area may reveal more organisms than the necrotic center.
- Bone marrow may be positive for trypanosomiasis and malaria when blood samples produce negative results. Bone marrow specimens are obtained through puncture of the sternum, iliac crest, vertebral processes, trochanter, or tibia.
- Puncture or biopsy samples from a lymph node may be examined for the presence of trypanosomiasis, leishmaniasis, toxoplasmosis, and filariasis.
- Mucous membrane lesion or skin samples may be obtained through scraping, needle aspiration, or biopsy.
- CSF may contain trypanosomes and Toxoplasma organisms.
- Sputum may reveal Paragonimus westermani (lung fluke) eggs. Occasionally, the larvae and hookworm of S. stercoralis or Ascaris lumbricoides may be expectorated during pulmonary migration. In pulmonary echinococcosis (hydatid disease), hydatid cyst contents may be found in sputum.
- Specimens taken from cutaneous ulcers should be aspirated below the ulcer bed rather than at the surface. A few drops of saline may be introduced by needle and syringe to aspirate intracellular leishmanial organisms.
- Corneal scrapings or biopsy specimens can be examined histologically or cultured for the presence of Acanthamoeba. This organism is rare but can cause keratitis among contact lens wearers.
- Films for blood parasites are usually prepared when the patient is admitted. Samples should be taken at 6- to 18-hour intervals for at least 3 successive days.

Diagnosis of Fungal Disease

Fungal diseases, also known as mycoses, are believed to be more common now than in the past because of increased use of antibacterial and immunosuppressive drugs (Table 7.6). Fungi prefer the debilitated host, the person with chronic disease or impaired immunity, or a patient who has been receiving prolonged antibiotic therapy.

Of more than 200,000 species of fungi, approximately 200 species are generally recognized as being pathogenic for humans. Fungi live in soil enriched by decaying nitrogenous matter and are capable of maintaining a separate existence through a parasitic cycle in humans or animals. The systemic mycoses are not communicable in the usual sense of human-to-human or animal-to-animal transfer. Humans become accidental hosts through inhalation of spores or by introduction of spores into tissues through trauma. Altered susceptibility may result in fungal lesions; this frequently occurs in patients who have a debilitating disease, diabetes, or impaired immunologic responses due to steroid or antimetabolite therapy. Prolonged administration of antibiotics can result in a fungal superinfection.

TABLE 7.6 Fungal Diseases and Their Laboratory Diagnosis

Disease	Causative Organism	Source of Specimen	Diagnostic Tests
Aspergillosis	Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus	Sputum, tissue, ear, corneal scraping	Smear and culture, serologic test, chest x-ray, computed tomography
Blastomycosis	Blastomyces dermatitidis	Skin lesion, sputum, bone, joint	Smear and culture, serologic test, skin biopsy, urine antigen test
Candidiasis	Candida albicans	Mucous membrane, sputum, blood, tissue, urine, CSF	Smear and culture
Coccidioidomycosis	Coccidioides immitis	Sputum, bone, skin, joint, CSF	Smear and culture, serologic skin test, biopsy
Cryptococcosis	Cryptococcus neoformans	CSF, sputum, urine	Smear and culture, serologic test, antigen detection
Histoplasmosis	Histoplasma capsulatum	Sputum, urine, blood, bone marrow	Smear and culture, serologic test, biopsy, urine antigen test
Mucormycosis	Members of order Mucorales (Absidia, Rhizopus, Mucor)	Nose, pharynx, stool, CSF, sputum, ear	Smear and culture, biopsy
Paracoccidioidomycosis	Paracoccidioides	Lung tissue, sputum, bone, CSF	Smear and culture, serologic test, biopsy
Pseudallescheriasis	Allescheria boydii	Lesions of skin, bone, brain, joint	Smear and culture, biopsy
Sporotrichosis	Sporothrix schenckii	Skin lesion, CSF, bone marrow, ear	Smear and culture, biopsy, serologic test
Tinea pedis (athlete’s foot)	Epidermophyton spp. and Candida albicans, Trichophyton mentagrophytes, Trichophyton rubrum	Skin	Skin scrapings for smear and culture
Tinea capitis (ringworm of scalp)	Microsporum (any spp.) and Trichophyton (all except T. concentricum)	Skin, hair	Hair, skin scrapings for smear and culture
Tinea barbae (ringworm of beard, barber’s itch)	Trichophyton and Microsporum spp.	Skin, hair	Hair, skin scrapings for smear and culture
Tinea cruris (jock itch)	Epidermophyton spp. and Candida albicans	Skin	Skin scrapings for smear and culture
Tinea corporis (ringworm of the body)	Trichophyton rubrum, Trichophyton tonsurans	Skin	Skin scrapings for smear and culture
Tinea unguium (nail)	Trichophyton rubrum, Trichophyton tonsurans, Trichophyton verrucosum, Epidermophyton spp.	Nail	Nail culture

Fungal diseases may be classified according to the type of tissues involved:

Dermatophytoses include superficial and cutaneous mycoses, such as athlete’s foot, ringworm, and “jock itch.” Species of Microsporum, Epidermophyton, and Trichophyton are the causative organisms.
Subcutaneous mycoses involve the subcutaneous tissues and muscles.
Systemic mycoses involve the deep tissues and organs and are the most serious of the three groups.

Amphotericin B, introduced into practice in 1958, was for many years the only drug available to treat invasive fungal infections. Now ketoconazole, fluconazole, itraconazole, and lipid formulations of amphotericin B provide alternative choices when treatment of fungal disease is warranted.

Collection of Hair and Skin Specimens

Clean the suspected area with 70% alcohol to remove bacteria. Use sterile techniques and standard precautions.
Scrape the peripheral erythematous margin of putative “ringworm” lesions with a sterile scalpel or wooden spatula and place the scrapings in a covered sterile container.
Clip samples of infected scalp or beard hair and place in a covered sterile container.
Pluck hair stubs out with tweezers because the fungus is usually found at the base of the hair shaft. Use of a Wood’s light in a darkened room helps identify the infected hairs.
Samples from infected nails should be procured from beneath the nail plate to obtain softened material from the nail bed. If this is not possible, collect shavings from the deeper portions of the nail and place them in a covered sterile container.

Common Diagnostic Methods for Fungal Diseases

A Wood’s light is used to determine presence of a fungus directly on hair. A Wood’s light is a lamp that uses ultraviolet rays of 3660A. In a darkened room, infected hairs fluoresce a bright yellowgreen under the Wood’s light.
Direct microscopic examination of tissue samples placed on a slide is performed to determine whether a fungus is actually present. The potassium hydroxide (KOH) test or Calcofluor white stain test is used to detect the presence of mycelial fragments, arthrospores, spherules, or budding yeast cells and involves mixing the specimen with the reagent on a glass slide. The slide is then microscopically examined for fungal elements.
A fluorescent brightener, Calcofluor white, fluoresces when exposed to ultraviolet light. This reagent stains the fungi, causing them to exhibit a fluorescence that can be detected microscopically. It can be used on tissue and has the same sensitivity as KOH. Moreover, it allows for easier and faster detection of fungal elements. Calcofluor white-stained specimens can also be examined under bright-field or phase-contrast microscopy.
Cultures are done to identify the specific type of fungus. Fungi are slow growing and are subject to overgrowth by contaminating and more rapidly growing organisms. Fungemia (fungus in the blood) is an opportunistic infection, and often, a blood culture reveals the earliest suggestion of the causative organism.
For fungal serology tests, single titers greater than 1:32 usually indicate the presence of disease. A fourfold or greater rise in titer of samples drawn 3 weeks apart is significant. However, serologic diagnosis of Candida and Aspergillus species can be disappointing. Complement fixation tests for histoplasmosis and coccidioidomycosis can aid in the diagnosis of these diseases. The immunodiffusion test is helpful for the diagnosis of blastomycosis.
Antigen tests performed on urine specimens are available for the detection of disseminated Histoplasma capsulatum and Blastomyces dermatitidis. The urinary antigen test has a 92% sensitivity.

Types of Specimens

Skin
Nails
Hair
Ulcer scrapings
Pus
CSF
Urine
Blood
Bone marrow
Stool
Bronchial washings
Tissue biopsy specimens
Prostatic secretions
Sputum

Diagnosis of Spirochetal Disease

Spirochetes appear as spiral and curved bacteria. The four genera of spiral and curved bacteria— Borrelia, Treponema, Leptospira, and Spirillum (Table 7.7)—include several human pathogens. Most spirochetes multiply within a living host. Pathogenic Treponema organisms are transmitted from person to person through direct contact. Borrelia pass through an arthropod vector. Leptospira are usually contracted accidentally by humans through water contaminated with animal urine or a bite by an infected animal.

TABLE 7.7 Spirochetal Diseases and Their Laboratory Diagnosis

Disease	Causative Organism	Source of Specimen	Diagnostic Tests
Pinta	Treponema carateum	Skin	Skin smear, serologic test
Rat-bite fever	Spirillum minor, Streptobacillus moniliformis	Blood, joint fluid, abscess	Culture, serology
Relapsing fever	Borrelia recurrentis	Blood	Blood smear
Syphilis	Treponema pallidum	Skin lesion	Skin smear, nonspecific treponemal (VDRL, RPR) and specific treponemal (FTA-ABS) serologic tests
Weil’s disease (leptospiral jaundice)	Leptospira interrogans	Urine, blood, CSF	Culture, serologic test
Yaws	Treponema pertenue	Skin	Culture, serologic test
Lyme disease	Borrelia burgdorferi	Skin lesion, blood, CSF	Serologic test
Nonvenereal syphilis	Treponema endemicum	Skin, blood	Serologic test

Clinical Considerations

Borrelia appear in the blood at the onset of relapsing fever. Louse-borne relapsing fever is caused by Borrelia recurrentis, tick-borne relapsing fever by several other Borrelia species, and Lyme disease by Borrelia burgdorferi. To date, there is no evidence that Lyme disease is transmitted by person-to-person contact.
Treponema (Borrelia) vincentii is the species responsible for ulcerative gingivitis (trench mouth).
- Treponema pallidum is the species responsible for venereal syphilis in humans.
- Treponema pallidum subsp. pertenue is the causative agent of yaws (an infectious nonvenereal disease).
- Treponema carateum causes pinta (carate).
- Treponema pallidum subsp. endemicum is the cause of endemic nonvenereal syphilis (bejel).
Leptospira is the genus of microorganism responsible for Weil’s disease (infectious jaundice), swamp fever, swineherd’s disease, and canicola fever.
- The organism is widely distributed in the infected person and appears in the blood early in the disease process.
- After 10 to 14 days, the organisms appear in considerable numbers in the urine.
- Patients with Weil’s disease show striking antibody responses; serologic testing is useful for diagnosis of this disease.
Streptobacillus moniliformis and Spirillum minor are the species responsible for rat-bite fever. Although this condition occurs worldwide and is common in Japan and Asia, it is uncommon in North and South America and most European countries. Cases in the United States have been linked to bites or scratches by infected rodents (such as rats, mice, and gerbils). The case fatality rate is 7-10% among untreated patients.

Reference Values

Normal

Negative enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay (IFA) for the antibody to Borrelia burgdorferi

Interventions

Pretest Patient Care

Explain purpose and procedure. Symptoms of Lyme disease include rash (erythema migrans), arthritis, facial paralysis, tingling or burning sensations in the extremities, and meningitis.
Follow Chapter 1 guidelines for safe, effective, informed pretest care.

Posttest Patient Care

Interpret test results; monitor and counsel patient appropriately. Recommended treatment of patients with Lyme disease includes antibiotics (e.g., amoxicillin, doxycycline, and cefuroxime axetil).
Follow Chapter 1 guidelines for safe, effective, informed posttest care.

Diagnosis of Viral and Mycoplasmal Infection

Viral infections are the most common of all human infections. Once thought to be confined to the childhood years, viral infections in adults have increasingly been recognized as the cause of significant morbidity and mortality. They also affect immunosuppressed and elderly patients (Chart 7.1). Viruses are responsible for hepatitis, AIDS, and other sexually transmitted infections (STIs).

Viruses are submicroscopic, filterable, infectious organisms that exist as intracellular parasites. They are divided into two groups according to the type of nucleic acid they contain: RNA or DNA.

The mycoplasmas are scotobacteria without cell walls that are surrounded by a single triple-layered membrane; they are also known as pleuropneumonia-like organisms (PPLOs). Physiologically, mycoplasmal infections are considered to be intermediate between those caused by bacteria and those
caused by rickettsiae. One species, Mycoplasma pneumoniae, is recognized as the causative agent of primary atypical pneumonia and bronchitis. Other species are suspected as possible causal agents for urethritis, infertility, early-term spontaneous abortion, rheumatoid arthritis, myringitis, and erythema multiforme.

CHART 7.1 Viral Infections in Infants, Children, and Adults

Infection or Syndrome	Suspected Viral Agents
Infants and Children
Upper respiratory tract infection	Rhinovirus, coronavirus, parainfluenza, adenovirus, respiratory syncytial virus, influenza, human metapneumovirus
Pharyngitis	Adenovirus, Coxsackie A, herpes simplex, Epstein-Barr, rhinovirus, parainfluenza, influenza
Croup	Parainfluenza, respiratory syncytial, human metapneumovirus
Bronchitis	Parainfluenza, respiratory syncytial, human metapneumovirus
Bronchiolitis	Respiratory syncytial, parainfluenza, human metapneumovirus
Pneumonia	Respiratory syncytial, adenovirus, influenza, parainfluenza
Gastroenteritis	Rotavirus, adenoviruses 40-41, calicivirus, astrovirus, Norovirus
Adults
Upper respiratory tract infection	Rhinovirus, coronavirus, adenovirus, influenza, parainfluenza
Pneumonia	Coxsackie B
Gastroenteritis	Norovirus
All Persons
Parotitis	Mumps, parainfluenza
Myocarditis/pericarditis	Coxsackie and echoviruses
Keratitis/conjunctivitis	Herpes simplex, varicella-zoster, adenovirus
Pleurodynia	Coxsackie B
Herpangina	Coxsackie A
Febrile illness with rash	Enteroviruses
Infectious mononucleosis	Epstein-Barr, cytomegalovirus
Meningitis	Enteroviruses, lymphocytic choriomeningitis, herpes simplex virus
Encephalitis	Herpes simplex, togaviruses, bunyaviruses, flaviviruses, rabies, enteroviruses, measles, human immunodeficiency virus (HIV), JC virus
Hepatitis	Hepatitis A, B, C, non-A, non-B; delta agent; E
Hemorrhagic cystitis	Adenovirus, BK virus
Cutaneous infection with rash	Herpes simplex, varicella-zoster, enteroviruses, Epstein-Barr, measles, rubella, parvovirus, human herpesvirus 6
Hemorrhagic fever	Ebola, Marburg, Lassa, hantavirus
Acute respiratory failure	Hantavirus

Viruses and mycoplasmas are infectious agents small enough to pass through bacteria-retaining filters. Although small size is the only property they have in common, viruses and mycoplasma cause illnesses that are often indistinguishable from each other in terms of clinical signs and symptoms; in addition, both frequently occur together as dual infections. Therefore, the serologic (antigen-antibody) procedures commonly used for diagnosing viral infection are also used for diagnosing mycoplasmal infections (Table 7.8).

Approach to Diagnosis

Isolation of the virus in tissue culture remains the gold standard for detection of many common viruses. Diagnostic modalities include the following:
- Tissue culture
- Direct detection in specimens
- Identification through specific cytopathic effect
- Use of immunofluorescence and immunoperoxidase, latex agglutination, or ELISA to identify
- Visualization through an electron microscope
- Direct nucleic acid hybridization probe and nucleic acid amplification assay
Serologic studies for antigen-antibody detection are valuable in regard to viral disease. Epstein-Barr virus (EBV) and human hepatitis viruses are routinely serodiagnosed. Classically, a fourfold rise in antibody titer is used to identify a particular infectious agent, provided that the pathogenesis of the agent agrees with the symptoms of the infected patient. An acute-phase serum is collected within the first several days after symptom onset. A convalescent-phase serum is collected 2 to 4 weeks later. A fourfold difference in antibody titer between the two sera is statistically significant. Alternatively, detection of specific immunoglobulin M (IgM) suggests acute infection. IgG antibody without IgM suggests infection sometime in the past.
Available cell cultures vary greatly in their sensitivity to different viruses. One cell type or species may be more sensitive than another for detecting the virus in low titers. For example, human embryonic kidney (HEK) or monkey kidney (1 MK) can be used for adenovirus, enterovirus, herpes simplex, measles, influenza, parainfluenza, and rubella; however, HEK cannot be used for cytomegalovirus (CMV) or influenza.
The critical first step in successful viral diagnosis is the timely and proper collection of specimens. The choice of which specimen to collect depends on typical signs and symptoms and the suspected virus. Improper specimen choice and collection is one of the biggest factors in diagnostic delays.

Specimen Collection

Collect specimens for viruses as early as possible during the course of the illness, preferably within the first 4 days after symptom onset. If specimen collection is delayed for 7 or more days after symptoms appear, diagnosis will be compromised. Virus titers are highest in the early part of the illness, when the host has not yet mounted a robust immune response. Little neutralizing antibody is present. Detection of a virus by culture, direct detection, or serology is greatly enhanced when the virus titers are high.

Sampling procedure

For localized infection:
- Direct sampling of affected site (e.g., throat swab, skin scraping)
- Indirect sampling. For example, if CSF is the target sample in a central nervous system infection, the indirect approach would involve obtaining throat or rectal swabs for culture.

Sampling from more than one site, for example, in disseminated infection or with nonspecific clinical findings

TABLE 7.8 Viral Infections and Their Laboratory Diagnosis

Only gold members can continue reading. Log In or Register to continue

Stay updated, free articles. Join our Telegram channel

Tags: Manual of Laboratory and Diagnostic Tests A

Jun 11, 2016 | Posted by drzezo in PATHOLOGY & LABORATORY MEDICINE | Comments Off

Full access? Get Clinical Tree

Get Clinical Tree app for offline access

Get Clinical Tree app for offline access

Infection Type and Virus Information		Throat	Stool/Rectal Swab	CSF	Urine	Vesicle Fluid/Swab	Conjunctival Swab/Scraping	Other	Blood Serology	Additional Information
Respiratory
	Adenovirus	X						Nasopharyngeal swab	Yes
	Enterovirus	X							No^*
	Herpes simplex virus (HSV)	X

Basicmedical Key

Fastest Basicmedical Insight Engine

Microbiologic Studies

Like this:

Related

Stay updated, free articles. Join our Telegram channel

Full access? Get Clinical Tree

Basicmedical Key

Fastest Basicmedical Insight Engine

Microbiologic Studies

Share this:

Like this:

Related

Related posts:

Stay updated, free articles. Join our Telegram channel

Full access? Get Clinical Tree