Microbiologic Studies

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



    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


    Cryptococcus neoformans

    Respiratory syncytial virus

    Influenza viruses

    Parainfluenza viruses




    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


    Campylobacter jejuni

    Clostridium botulinum

    Entamoeba histolytica

    Escherichia coli Salmonella spp.

    Shigella spp.

    Staphylococcus aureus

    Vibrio cholerae

    Vibrio comma

    Vibrio parahaemolyticus

    Yersinia enterocolitica

    Clostridium difficile


    Hepatitis A, B, and C

    Giardia lamblia Cryptosporidium spp.


    Aeromonas sp.

    Plesiomonas sp.



    Streptococcus agalactiae

    Escherichia coli, other


    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



    Bacteroides spp.

    Clostridium spp.


    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


    Listeria monocytogenes

    Streptococcus agalactiae

    (Group B)

    Staphylococcus spp.

    Escherichia coli

    Herpes simplex virus


    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


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


Causative Organism

Source of Specimen

Diagnostic Tests


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


Haemophilus ducreyi

Genital lesion biopsy

Lesion smear and culture; biopsy


Vibrio cholerae


Stool smear and culture


Chlamydia psittaci

Blood, sputum, lung tissue

Culture, smear, serologic tests


Corynebacterium diphtheriae


Nasopharyngeal smear and culture


Erysipelothrix rhusiopathiae

Lesion, blood

Smear and culture


Neisseria gonorrhoeae

Cervix, urethra, CSF, blood, joint fluid, throat

Smear and culture, nucleic acid amplification assay

Granuloma inguinale (donovanosis)

Calymmatobacterium granulomatis

Groin lesion


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


Serologic test

Legionnaires’ disease

Legionella pneumophila


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


Listeria monocytogenes

Blood, CSF, amniotic fluid, placenta, vagina

Smears and culture, serologic test


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


Clostridium tetani


Smear and culture

Toxic shock syndrome

Staphylococcus aureus




Mycobacterium tuberculosis

Sputum, gastric washings, urine, CSF

Smear and culture of sputum, gastric washings, urine, and CSF; skin test, QuantiFERON-TB assay


Francisella tularensis

Skin, lymph node, ulcer tissue biopsy, sputum, bone marrow

Serologic test, smear and culture


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


Nocardia asteroides

Sputum, lesion

Smear and culture


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.

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


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


    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


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


Geographic Distribution

Natural Cycle

Transmission to Humans

Serologic Diagnosis





Epidemic typhus*

Rickettsia prowazekii


Body louse


Infected louse feces into broken skin

Positive group- and typespecific serologic test

Endemic (murine) typhus

Rickettsia typhi




As above

Positive group- and typespecific serologic test

Spotted fever, Rocky Mountain spotted fever

Rickettsia rickettsii

Western hemisphere


Wild rodents, dogs

Tick bite

Serologic tests—IFA or latex agglutination

North Asian tickborne rickettsiosis

Rickettsia sibirica

Siberia, Mongolia


Wild rodents

Tick bite

Complement fixation

Boutonneuse fever

Rickettsia conorii

Africa, Europe, Mideast, India


Wild rodents

Tick bite

Positive group- and typespecific serologic test

Queensland tick typhus

Rickettsia australis



Marsupials, wild rodents

Tick bite

Complement fixation

Rickettsial pox

Rickettsia akari

North America, Europe


Mouse, other rodents

Mite bite


Scrub typhus

Rickettsia tsutsugamushi

Asia, Australia, Pacific Islands


Wild rodents

Mite bite

Specific complement fixation positive in about 50% of patients, and indirect immunofluorescence


Ehrlichia canis, Ehrlichia sennetsu

Southeast Asia



Tick bite

PCR amplification, peripheral blood smears

Q fever

Coxiella burnetii



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


Infected louse feces into broken skin

PCR, culture and serologic tests

Oroya fever

Bartonella bacilliformis

Peru, Ecuador, Columbia, Brazil

Sand fly


Bite of sand fly

PCR, culture, serologic tests

* Recurrence years after original attack of epidemic typhus.

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


    Causative Organism

    Source of Specimen

    Diagnostic Tests


    Entamoeba histolytica

    Stool, liver

    Stool smear, rectal biopsy, serologic test, antigen test


    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


    Ova and parasite examination, Scotch tape test for Enterobius vermicularis

    Chagas’ disease

    Trypanosoma cruzi

    Blood, spinal fluid

    Giemsa- or Wright-Giemsa-stained smear


    Cryptosporidium parvum

    Stool, lung, gallbladder

    Ova and parasite examination, antigen test, direct fluorescent antibody test


    Taenia solium larvae

    Muscle and brain

    Muscle and brain cyst biopsy, serology


    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


    Scotch tape test


    Wuchereria bancrofti, Brugia malayi, Loa loa


    Blood smear, lymph node biopsy, serologic test


    Giardia lamblia

    Stool, duodenal aspirate or biopsy

    Ova and parasite examination, antigen test, direct fluorescent antibody test, microscopic examination of Entero test


    Ancylostoma duodenale, Necator americanus


    Ova and parasite examination



    Isospora belli

    Leishmania donovani


    Liver, bone marrow, blood

    Ova and parasite examination

    Giemsa- or Wright-Giemsa-stained smear and culture, lymph node and spleen biopsy


    Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale

    Blood, bone marrow

    Giemsa- or Wright-Giemsa-stained smear


    Acanthamoeba culbertsoni

    CSF, corneal biopsy or scraping

    Smear and tissue culture


    Naegleria fowleri




    Sarcocystis hominis or Sarcocystis suihominis


    Ova and parasite examination


    Blastocystis hominis


    Ova and parasite examination


    Onchocerca volvulus


    Skin biopsy with histopathologic exam


    Paragonimus westermani

    Sputum, stool

    Ova and parasite examination, serologic test, skin test


    Sarcoptes scabiei


    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


    Strongyloides stercoralis

    Stool, duodenal aspirate

    Ova and parasite examination, serologic test


    Toxoplasma gondii

    Blood, tissue, CSF

    Serologic test, tissue smear, biopsy


    Trichinella spiralis


    Serologic test, skin test, muscle biopsy


    Trichomonas vaginalis

    Vagina, bladder, urethra

    Vaginal and urethral smear and culture, DNA probe


    Trichuris trichiura


    Ova and parasite examination


    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


    Serologic test, skin test, liver biopsy


    Fasciola hepatica, Clonorchis sinensis, Fasciolopsis buski


    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


Causative Organism

Source of Specimen

Diagnostic Tests


Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus

Sputum, tissue, ear, corneal scraping

Smear and culture, serologic test, chest x-ray, computed tomography


Blastomyces dermatitidis

Skin lesion, sputum, bone, joint

Smear and culture, serologic test, skin biopsy, urine antigen test


Candida albicans

Mucous membrane, sputum, blood, tissue, urine, CSF

Smear and culture


Coccidioides immitis

Sputum, bone, skin, joint, CSF

Smear and culture, serologic skin test, biopsy


Cryptococcus neoformans

CSF, sputum, urine

Smear and culture, serologic test, antigen detection


Histoplasma capsulatum

Sputum, urine, blood, bone marrow

Smear and culture, serologic test, biopsy, urine antigen test


Members of order Mucorales (Absidia, Rhizopus, Mucor)

Nose, pharynx, stool, CSF, sputum, ear

Smear and culture, biopsy



Lung tissue, sputum, bone, CSF

Smear and culture, serologic test, biopsy


Allescheria boydii

Lesions of skin, bone, brain, joint

Smear and culture, biopsy


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 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 scrapings for smear and culture

Tinea corporis

(ringworm of the body)

Trichophyton rubrum, Trichophyton tonsurans


Skin scrapings for smear and culture

Tinea unguium (nail)

Trichophyton rubrum, Trichophyton tonsurans, Trichophyton verrucosum, Epidermophyton spp.


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


Causative Organism

Source of Specimen

Diagnostic Tests


Treponema carateum


Skin smear, serologic test

Rat-bite fever

Spirillum minor, Streptobacillus moniliformis

Blood, joint fluid, abscess

Culture, serology

Relapsing fever

Borrelia recurrentis


Blood smear


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


Treponema pertenue


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


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

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.

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

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      Jun 11, 2016 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Microbiologic Studies

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      Infection Type and Virus Information


      Stool/Rectal Swab



      Vesicle Fluid/Swab

      Conjunctival Swab/Scraping


      Blood Serology

      Additional Information




      Nasopharyngeal swab





      Herpes simplex virus (HSV)