Microbiologic Studies



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 (Chart 7.1). Some organisms that were formerly considered insignificant contaminants or commensals have taken on roles as causative agents for opportunistic diseases in patients with HIV infection or other immunodeficiency syndromes or diseases associated with a compromised health status. 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









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

































      Nasopharyngeal and Oropharyngeal Specimens


      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 spp.


      Campylobacter jejuni


      Clostridium botulinum


      Entamoeba histolytica


      Escherichia coli


      Salmonella spp.


      Shigella spp.


      Staphylococcus aureus


      Vibrio cholerae


      Vibrio vulnificus


      Vibrio parahaemolyticus


      Yersinia enterocolitica


      Clostridium difficile


      Rotavirus


      Hepatitis A, B, and C


      Giardia lamblia


      Cryptosporidium spp.


      Norovirus


      Aeromonas sp.


      Plesiomonas sp.


      Leptospira spp.


      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 spp.


      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




    • Immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) antibody formation


    • Cell-mediated immune responses


  • Factors decreasing host resistance



    • Age (very young or very old)


    • 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; drugs (including legal, illegal, prescription, and nonprescription); 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 healthcare provider is responsible for collecting specimens for diagnostic examinations. Because procedures vary, check institutional protocols for specimen retrieval, transport, and 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. Polyestertipped swabs in a collection system containing an ampule of Stuart’s transport medium ensure adequacy of the specimen for 72 hours at room temperature.


  • Label specimen with the patient’s name, date, and test(s) ordered and place the specimen in a biohazard bag.




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 Part 173), 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.








OTHER CULTURES AND SMEARS




• Stool and Anal Cultures and Smears

Stool cultures are commonly done to identify bacteria associated with enteric infection. Of all specimens collected, feces are likely to contain the greatest number and greatest variety of organisms. For a routine stool culture, the stool is examined to detect and to rule out Salmonella, Shigella, Campylobacter, Aeromonas, Plesiomonas, and predominating numbers of Staphylococcus organisms; cultures for yeast, Pseudomonas, Yersinia, Vibrio, and Shiga toxin-producing E. coli have to be specifically requested, depending on laboratory practice. Clostridium difficile causes antibiotic-associated colitis. It is diagnosed by detection of the toxins.

A single negative stool culture should not be considered the end point in testing. At least three stool cultures collected on separate days are recommended if the patient’s clinical picture suggests bacterial involvement despite previous negative cultures. Moreover, once a positive diagnosis has been made, the patient’s personal contacts should also be tested to prevent a potential spread of infection.


Reference Values


Normal

The following organisms may be present in the stool of apparently healthy people:



  • C. albicans


  • Enterococcus spp.


  • E. coli


  • Proteus spp.


  • P. aeruginosa


  • Streptococcus spp.


  • Staphylococcus spp.




Interfering Factors

Stool from patients receiving barium, bismuth, mineral oil, or antibiotics is not satisfactory a specimen for identifying protozoa.



• Cerebrospinal Fluid (CSF) Cultures and Smears

Bacteriologic examination of CSF is an essential step in the diagnosis of any case of suspected meningitis. Acute bacterial meningitis is an infection of the meninges. It is a rapidly progressive, fatal infection if left untreated or if treated inadequately. Death can occur within hours of
symptom onset. Prompt identification of the causative agent is necessary for appropriate antibiotic therapy and aggressive treatment. Meningitis is caused by a variety of gram-positive and gramnegative microorganisms. Bacterial meningitis also can be secondary to infections in other areas of the body.

A smear and culture should be performed on all CSF specimens obtained from persons with suspected meningitis, whether the CSF appears clear (normal) or cloudy.

In bacterial meningitis (except TB meningitis), the CSF shows the following characteristics:



  • Purulence (usually)


  • Increased numbers of leukocytes


  • Preponderance of polymorphonuclear cells


  • Decreased CSF glucose concentration in relation to serum glucose


  • Elevated CSF protein concentration

In meningitis caused by the tubercle bacillus, viruses, fungi, or protozoa, the CSF shows the following characteristics:



  • Nonpurulent (usually)


  • Decreased mononuclear white cell count; increased lymphocytes


  • Normal or decreased CSF glucose concentration


  • Elevated CSF protein concentration

In those persons with suspected meningitis, the CSF is generally submitted for chemical and cytologic examinations as well as culture.


Indications for Collection



  • Viral meningitis


  • Pyogenic meningitis


  • TB meningitis


  • Chronic meningitis


Reference Values


Normal



  • Negative: no growth


  • Bacteria are not normally present in CSF. However, the specimen may be contaminated by normal skin flora during the process of CSF collection.






• Cervical, Urethral, Anal, and Oropharyngeal Cultures and Smears for Gonorrhea and Other Sexually Transmitted Infections (STIs)

These tests are done for patients with genital ulcers, vaginal lymphadenopathy, bacterial vaginosis (pathogens such as Gardnerella, Bacteroides, Prevotella, and Mobiluncus), lesions affecting epithelial surfaces, signs and symptoms of bacterial sexually transmitted infections (STIs), pelvic inflammatory disease, urethritis, or abnormal discharge and itching.


Reference Values


Normal

Negative: normal flora; negative for pathogenic antigens




• Tissue, Bone, and Body Fluid Cultures

Types of fluid collected for bacterial, viral, or fungal culture include pleural, ascitic, synovial, and pericardial fluid. Tissues may have to be minced or ground to release trapped bacteria before culturing.


Reference Values

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Sep 25, 2018 | Posted by in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Microbiologic Studies
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