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Clinical Microbiology Procedures Handbook, 3rd Edition
Editor: Lynne S. Garcia1
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Affiliations:
1: LSG & Associates, Santa Monica, California
Content Type: Reference
Format:
Electronic
Publication Year:
2010
Category: Clinical Microbiology
Print ISBN
:
9781555815271
e-ISBN
:
9781555817435
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
The collaborative efforts of over 150 experienced clinical microbiologists, medical laboratory technologists, and laboratory supervisors are included in the third edition of the Clinical Microbiology Procedures Handbook. This well-respected reference continues to serve as the sole major publication providing step-by-step protocols and descriptions that enable clinical microbiologists and laboratory staff personnel to perform all analyses, including appropriate quality control recommendations, from the receipt of the specimen through processing, testing, interpretation, presentation of the final report, and subsequent consultation.
In response to the ever-changing needs and responsibilities of the clinical microbiology field, most sections of the third edition have been extensively reviewed and updated. To accommodate the expanding role of clinical microbiologists, the new edition places greater emphasis on areas such as molecular approaches, bioterrorism, safety, and epidemiology/infection control in medical facilities. Procedures are formatted to adhere to the GP02-5A (2006) document of the National Committee for Clinical Laboratory Standards/Clinical and Laboratory Standards Institute (NCCLS/CLSI). Procedures are divided into preanalytical, analytical, and postanalytical considerations, with extensive discussion of each category. The icons in the margin of the text relate to safety recommendations, the use of standard precautions, a reminder for the user to record relevant reagent dates (receipt, opened, and expiration), as well as to reinforce quality control.
Electronic only, 2,540 pages, illustrations, index.
Book Summary
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VOLUME 1
Procedure Coding, Reimbursement, and Billing Compliance
Chapter 1.1 : Introduction
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When the first edition of this handbook was published, regulatory billing compliance for laboratory tests was not a major laboratory issue. Most hospital laboratories generally performed services for their own inpatients (and, occasionally, affiliated outpatients), and tests were billed according to a formula established by the business office. In the setting of prospective payment (e.g., diagnosis-related groups), payment credit was allocated based on a different formula. Today, however, microbiologists must be aware not only of the scientific basis of infectiousdisease diagnostics but also of the costing, coding, billing, and reimbursement for individual tests for patients seen in a broad spectrum of health care settings with coverage by an enormous number of health care plans. Jargon previously unknown in the clinical laboratory, such as reflex testing, upcoding, downcoding, local coverage decision (LCD), and national coverage decision (NCD), is now so extensive that a glossary of common terminology is included in this section ( Appendix 1.1-1 ). The goal is to be reimbursed adequately for all appropriate work performed in a manner that is in compliance with all regulations.
Chapter 1.2 : Procedure Coding, Reimbursement, and Billing Compliance
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The Office of the Inspector General (OIG) of the U.S. Department of Health and Human Services issued compliance program guidance for clinical laboratories in August 1998 ( 2 ). This anti-fraud and abuse document addresses Medicare and Medicaid program integrity, with emphasis on issues such as coding and billing; medical necessity; sales and marketing; arrangements with outside providers, suppliers, and vendors; and auditing and monitoring. Every clinical laboratory should be committed to doing business with any client, governmental or private, in an honest and trustworthy manner. While this document covers principles laid out in the federal compliance program guidance, the issues of integrity and the prevention of wrongdoing must be customized for each clinical laboratory to be compatible with the overall institutional compliance program. Specific details pertaining to elements of the Medicare program are detailed in Medicare Laboratory Payment Policy from the Institute of Medicine ( 1 ).
Specimen Collection, Transport, and Acceptability
Chapter 2.1 : Collection, Transport, and Manipulation of Clinical Specimens and Initial Laboratory Concerns
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The tables in this section address the initial events that lead to the accurate, rapid identification of microorganisms and viruses. Remember that results can only be as good as the original specimen. All the long-established precautions must be observed, keeping in mind that many specimens are obtained from anatomic sites that encourage specimen contamination with indigenous microbiota and results must be evaluated accordingly ( 10 ).
Aerobic Bacteriology
Chapter 3.1 : Introduction to the Section
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The Aerobic Bacteriology section of the handbook has been reorganized to place each part of the procedure together, including collection, specimen processing, supplies, QC, and the actual step-by-step testing. This will allow the user to see an overview of the entire procedure together. When several different methods of testing are acceptable, each option is presented. The users should not reproduce the procedural text of this handbook in its entirety but rather should choose among the various options presented to produce practical procedures applicable to their laboratory.
Chapter 3.2 : Staining Procedures
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The Gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and Gram reactions; it is additionally a critical test for the presumptive diagnosis of infectious agents and serves to assess the quality of clinical specimens ( 13 , 20 , 22 ). The test was originally developed by Christian Gram in 1884. The modification currently used for general bacteriology was developed by Hucker in 1921; it provides greater reagent stability and better differentiation of organisms. Other modifications have been specifically developed for staining anaerobes (Kopeloff's modification) and for weakly staining gram-negative organisms (Legionella spp., Campylobacter spp., Brucella spp., etc.) by using a carbol fuchsin or basic fuchsin counterstain ( 7 , 23 ). In fact, many laboratories use these counterstains routinely, especially for direct smears of clinical material.
Chapter 3.3 : Processing, Isolation, Detection, and Interpretation of Aerobic Bacteriology Cultures
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The initial processing of clinical specimens for bacteriology is a multifaceted endeavor involving a number of decision-making steps, including the need for processing the specimen for anaerobic bacteriology, mycology, virology, and parasitology, depending on the nature of the specimen. The need for direct tests, such as Gram stains, must also be considered. These issues will determine whether the specimen requires any pretreatment before inoculation.
Chapter 3.4 : Blood Cultures
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When bacteria or fungi overcome the host's normal defense mechanisms and enter the bloodstream through the lymphatics or from extravascular sites, they can quickly disseminate throughout the body, causing severe illness. In addition, the by-products of their metabolism can lead to septic shock, among the most serious complications of infectious diseases. Rapid recognition and immediate institution of appropriate treatment are essential. Laboratory diagnosis of bacteremia and fungemia depends on blood cultures, which are probably the most important cultures performed by the microbiology laboratory. Because the culture methods are so sensitive, the procedure must be carefully controlled beginning at the preanalytical stage (collection), to avoid the misinterpretation of a procurement-associated skin commensal microorganism as an agent of infection.
Chapter 3.5 : Body Fluid Cultures (Excluding Blood, Cerebrospinal Fluid, and Urine)
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Infection of normally sterile body fluids often results in severe morbidity and mortality; therefore, rapid and accurate microbiological assessment of these samples is important to successful patient management. Most organisms infecting these sites are not difficult to culture, but determining the significance of low numbers of commensal cutaneous microorganisms does present a challenge ( 14 ). With the increased use of prostheses, immunosuppressive therapeutic regimens, and long-term care of individuals with chronic debilitating disease, the likelihood of true infection with commensal organisms has increased, making accurate diagnoses difficult. Care must be taken during specimen collection and transport to ensure that the specimen is not contaminated. Any microorganism found in a normally sterile site must be considered significant, and all isolates must be reported.
Chapter 3.6 : Catheter Tip Cultures
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Intravascular (intra-arterial or intravenous) catheter insertions cause a break in the skin barrier amenable to infection. Table 3.6-1 lists the most common types of vascular and hemodialysis access catheters used for patient care that the laboratory may encounter. The continued presence of this foreign body predisposes further to infection, which can result from either colonization of the catheter by the cutaneous microbiota or, less commonly, hematogenous seeding due to hub contamination. Since infected catheters are usually exposed directly to sterile spaces, there is a risk that the infection will result in bacteremia. Intravascular catheter-related infections are a major cause of morbidity and mortality in the United States. The Infectious Disease Society of America practice guidelines for management of these infections include culture of both catheters and blood ( 9 ). Central catheter infection may manifest as infection at the skin insertion site, as cellulitis along the soft tissues overlying the tunneled portion, or as bacteremia without evidence of external infection at either of these superficial sites. Bacteremia occurs secondary to infection of the central catheter or as a manifestation of more serious complications, including septic thrombophlebitis or endocarditis. Laboratory confirmation of central catheter infection requires recovery of the same organism from a patient's blood and from cultures of the catheter tip and/or intracutaneous catheter segment. Clinical policy should instruct physicians to submit both catheter segments and blood cultures to the laboratory. The most common infecting organisms are Staphylococcus aureus,enterococci, Candidaspp., Pseudomonas aeruginosa, Enterobacteriaceae, and resident skin organisms, such as coagulase-negative staphylococci and Corynebacterium spp. The significance of this last group of organisms is not always clear, since the catheter is removed through the skin.
Chapter 3.7 : Cerebrospinal Fluid Cultures
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Bacterial meningitis is the result of infection of the meninges. Identification of the infecting agents is one of the most important functions of the diagnostic microbiology laboratory because acute meningitis is life-threatening. CSF from a patient suspected of meningitis is an emergency specimen that requires immediate processing to determine the etiologic agent.
Chapter 3.8 : Fecal and Other Gastrointestinal Cultures and Toxin Assays
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Gastroenteritis can be caused by bacteria, parasites, or viruses. With such a wide array of pathogens and the need for cost containment, physician input and practice guidelines ( 13 ) can help the laboratory determine which tests are appropriate for detecting the etiological agent of diarrhea. Microbiology laboratories should review the local epidemiology of bacterial enterocolitis and implement routine stool culture methods that will allow recovery and detection of all of the major pathogens causing most of the cases in their geographic area. All microbiology laboratories should routinely test for the presence of Salmonella spp., Shigella spp., and Campylobacter spp. on all stool cultures. Other major pathogens, such as Shiga-toxin-producing Escherichia coli, particularly E. coli O157 or enterohemorrhagic E. coli (EHEC), should also be routinely tested for on bloody stool samples during the spring, summer, and early fall months in geographic areas where the prevalence of these strains has been shown to be increased. Microbiology laboratories situated in or near coastal communities may also test for Aeromonas and Vibrio spp. since the prevalence of these types of infections is increased with exposure to water or contaminated food such as shellfish.
Chapter 3.9 : Genital Cultures
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Specimens from genital sites are sent to the clinical microbiology laboratory for detection of microorganisms from females presenting with clinical syndromes such as cervicitis, vulvovaginitis, urethritis, bacterial vaginosis (BV), salpingitis (pelvic inflammatory disease [PID]), endometritis, or genital ulcers and from males exhibiting urethritis, epididymitis, prostatitis, or genital ulcers ( Tables 3.9.1-1 to 3.9.1-3 ) ( 3 ). Specimens are also submitted from pregnant females to diagnose the presence of organisms that may cause disease in the neonate. Less commonly, specimens are sent from children and postmenopausal women ( 16 , 17 , 22 , 25 , 26 ).
Chapter 3.10 : Ocular Cultures
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Inflammatory eye conditions may be due to a variety of diseases, and microorganisms play a major role in both acute and chronic diseases ( Table 3.10-1 ). The detection of infectious agents depends on knowledge of the site of infection and the severity of the process, because a variety of organisms cause infections of the eye. Unlike the procedures with other specimen types, it may be important for the physician to inoculate culture media at the bedside rather than transport the specimen to the laboratory for processing. This procedure describes the clinical syndromes associated with bacterial infections of the eye, the organisms associated with these syndromes, and the procedure for isolation of these infectious agents. In addition to aerobic bacterial culture, Table 3.10-2 indicates the media to inoculate for anaerobic, fungal, and mycobacterial cultures. Refer to the respective sections of the handbook for workup of the microorganisms that are not covered in this procedure.
Chapter 3.11 : Respiratory Tract Cultures
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Specimens from the upper respiratory tract (throat specimens, nasopharyngeal swabs, nasal discharges) can be easily obtained but are contaminated with resident microbiota. In addition, many micro organisms present in the nares and throat are found in both the disease and the carrier states ( 2 , 3 , 4 ). It is estimated that 60% of children sporadically carry Streptococcus pneumoniae in their nasal passages by the age of 2years ( 2 ). Because of this contamination, these specimens often do not provide accurate, clinically useful information for diagnosis of bacterial respiratory infection caused by organisms such as S. pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. On the other hand, these specimens are useful for the diagnosis of specific pathogens, whose presence in symptomatic patients most often indicates disease (i.e., Streptococcus pyogenes, Bordetella pertussis, Corynebacterium diphtheriae, and respiratory viruses). Nasal cultures are also performed as a part of the infection control of hospitalized patients to detect carriage of oxacillin-resistant Staphylococcus aureus or as part of a staphylococcal outbreak. In the latter case, nasal carriage by hospital employees may also be important (procedure 13.17). However, culture of nasopharyngeal specimens to detect carriage of potential pathogens such as Neisseria meningitidis, S. pneumoniae, and H. influenzae should be discouraged. Since these pathogens are all part of the normal oropharyngeal flora, the clinical relevance of culturing them from this site cannot be determined. In addition, reporting of normal oropharyngeal flora from this site may result in the patient receiving an unnecessary course of antibiotic therapy, thus fostering the development of resistance. Antibiotic prophylaxis of individuals in close contact with a patient with meningococcemia should be directed by the CDC guidelines, and should not be withheld based on the nasopharyngeal culture result ( 1 ). Likewise, nasopharyngeal specimen cultures for yeast or mold colonization in otherwise healthy asymptomatic individuals should be discouraged for similar reasons. Nasopharyngeal cultures for the detection of either Aspergillus spp., Mucor spp.,or other fungi may be warranted for immunocompromised patients who are suspected of having an invasive infection.
Chapter 3.12 : Urine Cultures
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Urinary tract infections (UTI) account for seven million visits to physicians' offices and over one million hospital admissions per year. Epidemiological studies by E. H. Kass ( 10 ) have shown that bacterial counts of ≥105 CFU/ml for a pure culture of gram-negative bacilli were found to be associated with acute bacterial infections of the urinary tract. In females with dysuria and acute UTI, other investigators reported that 102 CFU/ml can be significant ( 8 , 9 , 17 ). For infants and catheterized patients, low counts also have been shown to be significant ( 6 , 18 , 19 ). Because of the importance of colony counts for infection, urine cultures are always performed with an accompanying colony count ( 2 ).
Chapter 3.13 : Wound Cultures
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A wide variety of microorganisms that reside on the skin and mucous membranes of the body, as well as those found in the environment, can cause skin and soft tissue infections. These organisms enter the body through breaks in the skin or mucous membranes, through wounds made by trauma or bites (exogenous) or as a complication of surgery or foreign-body implants (endogenous), or they can be spread through the vascular system (hematogenous).
Chapter 3.14 : Leptospira Culture
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Leptospirosis is a spirochetal zoonosis caused by the genus Leptospira. pathogenic Leptospira infects about 160 wild and domesticated mammalian species serovars occur within some species ( 3 , 9 , worldwide, which excrete the organism in their urine. The human disease is more commonly associated with occupations or recreational activities associated with direct skin or mucous membrane contact either with the animal reservoir or with water, soil, or sewage contaminated with the animal urine. The genus Leptospira was previously divided into two species, Leptospira interrogans and Leptospira biflexa, which were differentiated by a number of biochemical tests ( 9 , 10 , 15 ). Most reports of human infection are based on this phenotypic classification system, and L. interrogans comprised all pathogenic strains, while L. biflexa was thought to include only saprophytic environmental strains ( 15 ). Leptospires are also assigned a serovar based on agglutination after cross-absorption with homologous antigen ( 8 . 10 ). L. interrogans comprises 218 serovars, while L. biflexa comprises more than 60 serovars ( 8 , 10 ). Leptospira serovars that are antigenically related are further grouped into serogroups, primarily for epidemiological purposes when interpreting serologic test results to distinguish closely related species.
Chapter 3.15 : Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma Cultures from Clinical Specimens
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Mycoplasma pneumoniae is a common cause of upper and lower respiratory infections in persons of all ages. Tracheobronchitis is the most common clinical syndrome, but pneumonia commonly occurs and extragenital infections have been described. A review of human disease caused by M. pneumoniae ( 5 ) contains more detailed information about clinical aspects of infections due to this organism.
Chapter 3.16 : Guidelines for Biochemical Identification of Aerobic Bacteria
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On the following pages are 48 biochemical procedures in alphabetical order, including both simple, rapid test procedures and standard conventional methods, for the identification of gram-positive and gram-negative bacteria. The biochemical tests have been selected that are most useful to laboratories, with emphasis on rapid testing. The list of tests includes the generally accepted tests that all laboratories should be able to perform to identify the clinically important microorganisms encountered in the laboratory, as well as some for use by reference or referral laboratories. Smaller laboratories may choose to perform fewer tests and refer cultures when less common microorganisms are found in culture. When identification to the species level is not clinically important, tests to separate these species are not included. When there is a choice of different tests that can be performed, both are presented and the user can choose which is preferable for use in the laboratory.
Chapter 3.17 : Biochemical Tests for the Identification of Aerobic Bacteria
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Acetamide agar is used to test an organism's ability to utilize acetamide by deamidation. The medium contains acetamide as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth is indicative of a positive test for acetamide utilization. When the bacterium metabolizes acetamide by the enzymatic action of an acylamidase, the ammonium salts are broken down to ammonia, which increases alkalinity. The shift in pH turns the bromthymol blue indicator in the medium from green to blue, indicative of a positive test. Assimilation of acetamide will result in a yellow color and should not by mistaken for a positive result ( 2 ). In general, deamidation is limited to only a few organisms. This medium is recommended for differentiating Pseudomonas aeruginosa from other non-glucose-fermenting, gram-negative rods.
Chapter 3.18 : Schemes for Identification of Aerobic Bacteria
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Unlike gram-negative rods, it can be very difficult to sort out the identification of gram-positive cocci and rods. Many kits for staphylococcal identification have proved to be less sensitive than desired, and new DNA studies indicate that we have misidentified many streptococci. Gram-positive rods have been difficult to identify because there are hundreds of named species and thousands of genotypes or biochemical variants found in the environment and the normal microbiota of the human body, including skin, mucosal membranes, oropharynx, and genitourinary and gastrointestinal tracts. Thus, it is not within the scope of this handbook to identify all isolates, but to detect and identify the known pathogenic microorganisms in the human biosphere and to limit other identifications to those bacteria that are involved in disease from invasively collected specimens. The figures and tables that follow are designed to rapidly determine the agents of infection and to provide guidance for when to perform a kit identification or pursue other microorganisms. Because of the increasing microbial diversity and emergence of common pathogens having rare or unique phenotypic characteristics and the identification of new pathogens with incompletely defined phenotypes, more laboratories are relying on a combination of phenotypic and genotypic methods to report an accurate identification of many bacteria, so indications where molecular identification using DNA target sequencing may be useful are included ( 8 , 32 ).
Anaerobic Bacteriology
Chapter 4.1 : Introduction
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Anaerobic bacteria are a significant component of the normal microbiota of the human host. There are anaerobes present on most body surfaces and mucous membranes; they exist in large numbers throughout the entire gastrointestinal tract, from the mouth to the colon, with the exception of the stomach and esophagus; they are found in large numbers in the female genitourinary tract. In most areas, a true symbiotic relationship exists: humans supply the environment for the anaerobes to live and multiply in the presence of food, water, and a “friendly” atmosphere; the bacteria aid in digestion of foodstuffs for metabolism, prevent attachment of more virulent microbes by virtue of their presence in very large numbers, and make up a major component of the innate immunity of the host. In addition, the normal anaerobic microbiota bacteria are important in supplying needed vitamins and cofactors like vitamin K that humans cannot manufacture on their own ( 6 ).
Chapter 4.2 : Collection and Transport of Clinical Specimens for Anaerobic Culture
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Proper collection of specimens to avoid contamination with organisms of the normal microbiota and prompt transport to the laboratory for processing are essential. The isolation of anaerobic bacteria from appropriately collected clinical samples and reporting of that information as quickly as possible are extremely important for the clinician to be able to implement appropriate therapeutics. Gram stains are needed to provide a rapid analysis of the appropriateness of the sample and are essential for the correlation to what grows out in the laboratory.
Chapter 4.3 : Culture Media for Anaerobes
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The choice of media for use in the anaerobic bacteriology laboratory is important for the success of anaerobic bacteriology. The media must contain appropriate nutrients and supplements needed by clinically significant anaerobes. A combination of enriched, nonselective, selective, and differential media should be used for the initial processing, isolation, and presumptive identification of anaerobic bacteria from clinical specimens ( Fig. 4.3-1 and Tables 4.3-1 and 4.3-2 ) ( 1 - 3 ). Anaerobes have a wide range of nutritional needs; most, however, require hemin and vitamin K. Some studies suggest that freshly pre-pared, properly stored, highly enriched media are essential for recovery of anaerobes ( 4 , 8 ), while another study has shown that prereduced anaerobically sterilized (PRAS) media best support the growth of anaerobes ( 7 ). Recent studies have suggested that using media containing oxyrase may be another alternative ( 9 , 10 ). Media that have been exposed to air contain oxidized products that may delay or inhibit the growth of many anaerobes. The ideal media for use in anaerobic bacteriology, therefore, are those that have had limited exposure to oxygen.
Chapter 4.4 : Examination of Primary Culture Plates for Anaerobic Bacteria
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The goal of processing primary culture plates is to isolate significant anaerobic organisms present in the original specimen for identification and when susceptibility testing is indicated. The original Gram stain of the specimen is critical. At that time all morphological types should be carefully described and recorded. When evaluating culture plates, all morphotypes observed in the original Gram stain should match.
Chapter 4.5 : Incubation Techniques for Anaerobic Bacteriology Specimens
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This procedure describes the various environmental methods and incubation conditions used for anaerobic bacteriology specimens once a properly selected, collected, and transported specimen arrives in the laboratory. Ideally, the specimen is processed immediately on arrival in the laboratory and incubated under anaerobic conditions to prevent further exposure to oxygen. Refer to procedure 4.2 for collection procedures, to procedure 4.3 for an-aerobic media, and to procedure 4.4 for processing and inoculation techniques.
Chapter 4.6 : Rapid Disk, Spot Tests, and Other Methods for the Identification of Anaerobes
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Rapid disk, spot tests, and other methods described in this procedure provide a cost-effective system for the identification of anaerobes. Many of the tests described cost less than $0.25 each to perform (see Appendix 4.6-1 (turn to 4.6.13) for a summary of tests used for the rapid identification of anaerobes).
Chapter 4.7 : Commercial Kit Overnight Biochemical Systems for the Identification of Anaerobes
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Biochemical systems for identification of an anaerobe rely on the metabolic break-down of substrates and the production of end products during the growth of the isolated organism. There are at least two commercially packaged kit systems that fit this method for the identification of commonly isolated anaerobic bacteria in the clinical microbiology laboratory when species identification is needed: the API 20A (bioMérieux, Inc.) and the AN Microplate (Biolog) ( 1 – 6 ) ( Table 4.7-1 ). Both rely on overnight incubation of the test card before reading. The API 20A is manually filled and read; the AN Microplate is automatically filled but manually (visually) read. The API 20A uses 16 carbohydrates; its indicator system is bromcresol purple that turns yellow at a pH of 6.8 when a positive reaction occurs. The AN Microplate has a database of 361 anaerobes that are exposed to 95 preselected carbon sources for specific identification. The Minitek (BD Biosciences) kit was described in the last two editions of this handbook, but there were no references on the BD website that indicated that these systems were being manufactured any longer. Procedure 4.8 describes the BD Crystal ANR ID, which had largely taken over the role of the older Minitek panels.
Chapter 4.8 : Rapid Enzymatic Systems for the Identification of Anaerobes
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Rapid identification of anaerobes can be accomplished with commercially available microsystems that detect preformed enzymes within a few hours, eliminating the need for growth of the isolates ( 1 – 4 , 6 – 9 , 12 – 14 , 16 ). The systems that are available are listed in Table 4.8-1 . In a study that recently surveyed what methods laboratories were using to identify isolated anaerobes, 66% reported using a system that detected preformed enzymes ( 5 ). The systems listed in Table 4.8-1 require only 4 h of aerobic incubation after inoculation with a turbid suspension of the organism equivalent to a no. 3 or 4 McFarland standard. There are differences in the numbers and types of organisms included in each database as well as in the substrates that are used for differentiation. Some use fluorogenic and some use chromogenic substrates. Most require some reagent addition after incubation, before reading. Database and numerical identification profiles are provided for each system.
Chapter 4.9 : Rapid Biochemical Tests (4 Hours or Less) for the Identification of Anaerobes
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Many anaerobic isolates may be identified to the genus and species levels using a variety of preformed-enzyme tests or other rapid biochemical tests. In some in-stances, tests for identification beyond the level that can be attained with the various spot tests described in procedure 4.6 are needed. Presumptive or definitive identification of anaerobes is possible by using individual biochemical tests or a combination of preformed enzymatic manifestations.
Chapter 4.10 : Anaerobic Gram-Negative Bacilli
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The anaerobic gram-negative bacteria are part of the microbiota of the oral cavity, gastrointestinal tract, and urogenital tract ( 3 , 6 , 12 ). Many taxonomic changes have been instituted based on newer biochemical and molecular sequencing considerations. There are new genera and species for “old names” and newly described organisms, and these will only increase in numbers as better culture techniques and more advanced methods for molecular identification are utilized ( 3 - 13 , 15 , 17 , 20 , 21 ). These changes have made under-standing current taxonomy and correlating old and new names in the clinical literature more difficult for laboratorians and clinicians alike. Clinical decisions still require accurate, timely identification of clinically relevant anaerobic pathogens by the clinical microbiology laboratory. We, as laboratorians, need to keep abreast of the changes and relay those changes as efficiently as possible to our colleagues in a manner that maintains the old and new; i.e., always consider sharing with the clinician the old and new genera, for example, so that they can associate the two. Table 4.10-1 gives information about some of the changes in taxonomy of the anaerobic gram-negative bacilli.
Chapter 4.11 : Anaerobic Gram-Positive Bacilli
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Anaerobic gram-positive bacilli of clinical relevance in human infections are divided into two distinct groups: members of the genus Clostridium, which are spore-forming gram-positive anaerobic bacilli, and a group composed of more than 34 genera of non-spore-forming anaerobic gram-positive bacilli. Of the non-spore-formers, 6 genera are most commonly associated with clinical infections: Actinomyces, Bifidobacterium, Eubacterium, Eggerthella, Lactobacillus, and Propionibacterium ( 11 - 13 ). Two additional genera, Mobiluncus and Atopobium, have more recently been found in association with bacterial vaginosis and other infections; however, they are not easily recovered, and their pathogenicity is not as well understood ( 4 , 12 , 13 ). There have been many taxonomic changes among the an-aerobic gram-positive non-spore-forming bacilli. Some of these species that have been found in clinical samples are listed in Table 4.11-1 . Many of the anaerobic gram-positive bacilli are part of the normal microbiota of the oral cavity, gastrointestinal tract, genitourinary tract, and skin. They can, however, be associated with skin and soft tissue infections, periodontitis and other oral infections, pulmonary infections (usually in combination with other aerobes and anaerobes), genitourinary tract infections, and, in the case of Propionibacterium acnes, infected CSF shunts. Many of the non-sporeformers can be resistant to metronidazole, an antimicrobial agent that is usually effective against most other anaerobes.
Chapter 4.12 : Anaerobic Cocci
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The anaerobic cocci are a prominent part of the normal human microbiota of the skin, gastrointestinal tract, oral cavity, upper respiratory tract, and female genital tract. Anaerobic gram-positive cocci can be important human pathogens, and next to the anaerobic gram-negative bacilli, these anaerobes are the most commonly isolated anaerobes in clinically relevant infections ( 3 – 5 , 9 ). In four surveys of human anaerobic pathogens in various clinical specimens, anaerobic cocci constituted 24 to 31% of all isolates ( 3 , 9 ). The types of infections in which anaerobic gram-positive cocci predominate include infections in patients with a variety of head and neck infections, including periodontitis, chronic otitis media, chronic sinusitis, and brain abscesses; infectious processes in the female genital tract, including tubo-ovarian abscesses; and infections of the abdominal cavity, including peritonitis and perforated appendices. Many of these infections are polymicrobic, including other aerobes and anaerobes ( 3 – 5 , 9 ). Anaerobic gram-negative cocci, although not infrequently encountered in the clinical laboratory from clinical specimens, are not often associated with a great number of infections ( 4 , 5 ).
Chapter 4.13 : Suggestions for a Practical Scheme for the Workup of Anaerobic Cultures
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When a specimen is sent to the laboratory in an appropriate container and from an appropriate site with a request for anaerobe testing, a Gram stain should be performed and reported. The results of the Gram stain should direct the culture workup to be done as well as provide information to the clinician and the laboratory about the quality of the specimen and the potential for mixed aerobic and anaerobic floras. All anaerobe requests should receive a simultaneous aerobic culture, and these should be worked up in concert before the report is finalized. The scheme for identification of anaerobes can take on many models, depending upon the extent of workup required by the clinicians and the amount of expertise and time that can be allotted to their identification by the clinical microbiologist. Certainly, all single isolates from sterile sites should be completely worked up to genus and species name as much as is possible. It is less consistent among laboratories as to how much is fully identified in nonsterile sites or what should be identified when a “sterile” site, such as ascites fluid or abscess or even tissues, contains a mixed flora, including aerobic and anaerobic organisms, in a quantity of more than three organisms.
Chapter 4.14 : Clostridium difficile as a Pathogen Involved in Antimicrobial Agent-Associated Diarrhea, Colitis, and Pseudomembranous Colitis
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Clostridium difficile is the major cause of nosocomial diarrhea and the primary pathogen responsible for pseudomembranous colitis. In the United States, there are >300,000 cases per year of antimicrobial agent-associated diarrhea, colitis, or pseudomembranous colitis caused by this organism and its toxins. In a recent review article, one hospital reported that medical expenditures associated with C. difficile-associated disease were almost $1,000,000 per year ( 11 ). C. difficile can also be a rare cause of abscesses, wound infections, osteomyelitis, pleuritis, peritonitis, septicemia, and urogenital tract infections.
VOLUME 2
5 Antimicrobial Susceptibility Testing
Chapter 5.1 : Disk Diffusion Test
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A standardized inoculum of bacteria is swabbed onto the surface of a Mueller-Hinton agar (MHA) plate. Filter paper disks impregnated with antimicrobial agents are placed on the agar. After over-night incubation, the diameter of the zone of inhibition is measured around each disk. By referring to the tables in the NCCLS disk diffusion standard ( 1 ), a qualitative report of susceptible, intermediate, or resistant is obtained.
Chapter 5.2 : Broth Microdilution MIC Test
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The broth microdilution MIC method is used to measure (semiquantitatively) the in vitro activity of an antimicrobial agent against a bacterial isolate. A sterile plastic tray containing various concentrations of antimicrobial agents is inoculated with a standardized number of test bacteria. After overnight incubation at 35°C, the MIC is determined by observing the lowest concentration of an antimicrobial agent which will inhibit visible growth of the bacterium. MICs obtained are interpreted as susceptible, intermediate, or resistant, based on the criteria listed in the NCCLS MIC standard ( 1 ).
Chapter 5.3 : Beta-Lactamase Tests
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Beta-lactamases are enzymes produced by many clinically significant bacteria and are major mediators of bacterial resistance to beta-lactam agents. Routine beta-lactamase tests are based on visual detection of the end products of beta-lactamase hydrolysis, which is demonstrated with a colorimetric reaction. These tests primarily include the chromogenic cephalosporin method, the acidimetric method, and the iodometric method and are summarized in Appendix 5.3-1 . Not every method is satisfactory for detecting beta-lactamase produced by all of the bacteria for which the test is useful.
Chapter 5.4 : Oxacillin Salt-Agar Screen Test To Detect Oxacillin (Methicillin)-Resistant Staphylococcus aureus
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A standard number of bacteria is inoculated onto Mueller-Hinton agar (MHA) containing 6 µg of oxacillin per ml and 4% NaCl. Following overnight incubation, the appearance of growth indicates that the Staphylococcus aureus isolate is resistant to oxacillin and other penicillinase-stable penicillins (methicillin, nafcillin, cloxacillin, and dicloxacillin) ( 1 ).
Chapter 5.5 : Screen Tests To Detect High-Level Aminoglycoside Resistance in Enterococcus spp.
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High-level aminoglycoside resistance (HLAR) in enterococci is most commonly detected by assessing growth at high concentations of gentamicin (500 µg/ml) and sometimes streptomycin (1,000 µg/ml in broth and 2,000 µg/ml in agar) ( 1 ). Strains that show HLAR to gentamicin will not be synergistically killed with combinations of cell wall-active drugs (generally ampicillin, penicillin, or vancomycin) plus gentamicin. HLAR to gentamicin also means HLAR to tobramycin, netilmicin, amikacin, and kanamycin. HLAR to streptomycin equals resistance to combinations of cell wall-active drugs and streptomycin. See additional information in Appendix 5.5-1 . The agar screen test is described in detail here, and reference is made to the disk diffusion and the broth microdilution MIC test procedures for detecting HLAR.
Chapter 5.6 : Agar Screen Test To Detect Vancomycin Resistance in Enterococcus spp.
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The vancomycin agar screen is used to detect vancomycin-resistant enterococcal colonies that have been isolated from clinical or surveillance cultures. A standard number of bacteria is inoculated onto BHI agar containing 6 µg of vancomycin per ml. Following incubation, the appearance of growth indicates that the enterococcal isolate is likely to be resistant to vancomycin. MIC and species identification tests are subsequently required to determine if the isolate is a true vancomycin-resistant enterococcus (VRE) ( 1 , 2 ).
Chapter 5.7 : Broth Microdilution MIC Test for Anaerobic Bacteria
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The broth microdilution MIC method can be used to measure (semiquantitatively) the in vitro activity of an antimicrobial agent against an anaerobic bacterial isolate. This procedure is very similar to the broth microdilution method used for aerobic bacteria. Various concentrations of antimicrobial agents are dispensed into a multiwell plastic tray and inoculated with a test isolate. After 48 h of incubation at 35°C in an anaerobic environment, the MIC is determined by observing the lowest concentration of an antimicrobial agent which will inhibit visible growth of the test bacterium. MICs obtained are interpreted as susceptible, intermediate, or resistant, based on criteria defined by the NCCLS ( 1 ).
Chapter 5.8 : Etest
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A standardized inoculum of bacteria is swabbed onto the surface of a Mueller-Hinton agar (MHA) plate. Etest strips containing a continuous gradient of antimicrobial concentrations are placed on the agar surface. After overnight incubation, an elliptical zone of inhibition forms as the antimicrobial agent inhibits growth. The MIC is read where growth intersects the Etest strip. MICs can be interpreted as susceptible, intermediate, or resistant based on the tables in the NCCLS MIC standard ( 1 ).
Chapter 5.9 : Agar Dilution MIC Test for Anaerobic Bacteria
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The anaerobe agar dilution MIC method is a semiquantitative method for the determination of in vitro activity of an antimicrobial agent against anaerobic bacterial isolates. A series of agar plates, each containing a unique concentration of an antimicrobial agent, are inoculated with up to 36 isolates. After 48 h of incubation at 35°C in an anaerobic environment, the MIC is determined by observing the lowest concentration of antimicrobial agent which will inhibit visible growth of the test isolate. MICs obtained are interpreted as susceptible, intermediate, or resistant based on criteria defined by the NCCLS ( 2 ).
Chapter 5.10 : Tests to Assess Bactericidal Activity
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The minimum bactericidal concentration (MBC) test can be used to assess the ability of an antimicrobial agent to kill a bacterial isolate. MBC tests are performed after consultation in very select clinical situations when it is necessary to deter-mine the bactericidal activity of an anti-microbial agent against a bacterial isolate. These situations include those in which immune mechanisms offer little help in eradicating the infecting organisms, such as in patients with endocarditis or osteomyelitis or in immunosuppressed patients, particularly those with neutropenia. Bactericidal tests provide a rough prediction of bacterial eradication, but other factors may also impact the bacteriologic responses of patients. MBC testing is an accepted parameter in the evaluation of new antimicrobial agents and is frequently used as a research tool.
Chapter 5.11 : Serum Inhibitory and Bactericidal Titers
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Schlichter and MacLean first used serum inhibitory titers (SIT) to assess the effectiveness of penicillin in the treatment of subacute bacterial endocarditis ( 2 ). Other recognized clinical indications for obtaining serum bactericidal titers (SBT) to monitor effective therapy are osteomyelitis, closed-space infections such as meningitis or joint infection, and the use of oral antimicrobial agents after intravenous therapy. Controversy surrounds and will continue to surround the methodology, and there are few clinical situations in which the test is indicated.
Chapter 5.12 : Synergism Testing: Broth Microdilution Checkerboard and Broth Macrodilution Methods
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Microbiologists commonly use a two-dimensional, two-agent broth microdilution checkerboard to evaluate combinations of antimicrobial agents against microorganisms. A broth macrodilution limited series, agar dilution method, or disk diffusion method can also be used. Test methods are based on NCCLS broth dilution susceptibility methods ( 5 ) for evaluating the inhibitory or bactericidal activity ( 4 ) of specific concentrations in combination at a fixed time. In vitro interactions are calculated algebraically and interpreted as synergistic, indifferent, or antagonistic depending on whether the antibacterial activity of the combination is greater than, equivalent to, or less than, respectively, the activities of the individual agents. In vitro interactions can also be represented geometrically with isobolograms ( 1 ).
Chapter 5.13 : Quality Assurance Measures for Antimicrobial Susceptibility Testing
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QC is performed to ensure proper performance of antimicrobial susceptibility tests in order to provide accurate, reproducible, and timely results. The basic QC procedure used in clinical laboratories involves testing reference strains that have defined characteristics of susceptibility to the antimicrobial agent(s) tested. These strains must be properly maintained in order to ensure their reliable performance. Testing the QC strains controls many parameters of the antimicrobial susceptibility test; however, testing the reference strains alone does not always ensure reliable results when testing patients' isolates. Inclusion of supplemental control strains (particularly if recommended in the manufacturer's instructions when using a commercial test system), evaluation of susceptibility profiles on patients' isolates, verifying technologist competency, and review of cumulative susceptibility statistics are some of the additional measures that can be taken to further QC antimicrobial susceptibility tests.
Chapter 5.14 : Preparation of Routine Media and Reagents Used in Antimicrobial Susceptibility Testing
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McFarland turbidity standards are used to standardize the approximate number of bacteria in a liquid suspension by visually comparing the turbidity of a test suspension with the turbidity of a McFarland standard. McFarland standards are prepared by adding barium chloride to sulfuric acid to obtain a barium sulfate precipitate. By adjusting the volumes of these two reagents, standards of varying degrees of turbidity can be prepared to represent several different concentrations of bacteria. The standard most commonly used in the clinical microbiology laboratory for routine antimicrobial susceptibility tests ( 1 , 2 ) is 0.5, which represents 1.5 × 108 (generally, range is 1.0 × 108 to 2.0 × 108) bacteria/ml. McFarland standards are commercially available from several sources. As an alternative to the traditional barium sulfate standards, McFarland standards prepared from latex particles have recently become commercially available.
Chapter 5.15 : Preparation of Broth Microdilution MIC Trays
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Broth microdilution MIC trays may be prepared in-house for performance of quantitative antimicrobial susceptibility studies. If large numbers of trays are required, dilutions of the antimicrobial agents are made in large volumes and generally dispensed in 0.1-ml amounts into microdilution trays by using a commercial dispensing apparatus. For individual test trays, dilutions of the required antimicrobial agents may be accomplished with an eight-channel micropipette. The procedure described here is based on 96-well trays containing 0.1 ml per well. Not every aspect of broth microdilution tray preparation has been standardized, and for some steps, there are multiple ways of accomplishing identical results. The method presented here conforms closely to that described by the NCCLS ( 2 ) and is the procedure that has been successfully used with a Quick Spense (Sandy Springs In strument Co.) dispenser at the clinical microbiology laboratories of the University of California—Los Angeles for nearly two decades. It assumes that the layout of the tray in terms of positioning respective concentrations of each antimicrobial agent has been previously determined. The method is detailed for preparation of approximately 1,900 trays, but recommendations for preparing fewer trays are included.
Chapter 5.16 : Selecting Antimicrobial Agents for Testing and Reporting
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Developing protocols for antimicrobial susceptibility testing and reporting is best done with input from the infectious disease service, infection control, and the pharmacy and therapeutics committee. The goals are to provide clinically relevant information that will support cost-effective utilization of antimicrobial agents and to avoid reporting results that may adversely affect patient care. Because of differences in hospital formularies and laboratory functions, it is impossible to make specific recommendations and the suggestions described herein can only serve as a guide to the decision making processes. A list of commonly used antibacterial agents is shown in Appendix 5.16-1 .
Chapter 5.17 : Evaluating Antimicrobial Susceptibility Test Systems
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A wide variety of antimicrobial susceptibility test systems are available to microbiologists today. This procedure provides guidance on evaluating and choosing a system or method appropriate for your laboratory. The primary focus should be on determination of the systems' capabilities, assessment of costs and work flow, and examination of performance data.
Chapter 5.18 : Appendixes
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The list below is intended to provide guidance in acquiring materials for performing antimicrobial susceptibility tests as described in section 5. See procedure 3.1 for a listing of companies that supply media, reagents, QC strains, and other products necessary to perform the procedures.
6 Aerobic Actinomycetes
Chapter 6.1 : Specimen Examination and Primary Isolation
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Aerobic actinomycetes cause a variety of infections in humans: respiratory, cutaneous (such as mycetoma, lymphocutaneous and superficial skin [abscess or cellulitis] infections, or secondary cutaneous involvement with disseminated disease), and disseminated, with a marked tropism to the central nervous system, from a primary pulmonary infection. See Appendix 6.3.1–1 at the end of this section for a list of etiologic agents and the sites most commonly associated with these infections.
Chapter 6.2 : Media and Methods Used for the Identification of Aerobic Actinomycetes
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After initial isolation, subcultures must be incubated at 25, 35, and 45°C to determine the optimal temperature for growth of the microorganisms. Most isolates of Nocardia asteroides grow best at 35°C, while most isolates of Streptomyces grow best at 25°C. Any specimen suspected of containing a thermophilic actinomycete must also be incubated at 50°C, since all thermophiles grow at this elevated temperature. The actinomycetes can be identified to the genus level on the basis of a variety of conventional phenotypic characteristics (microscopic [Gram and acid-fast staining] and macroscopic morphologies, growth requirements, metabolism of glucose, arylsulfatase production, and growth in lysozyme) and chemotaxonomic characteristics (whole-cell isomers of diaminopimelic acid and mycolic acid composition) ( Table 6.1-1 , above). Some of these tests are beyond the capabilities of most clinical laboratories. However, both microscopic and macroscopic morphologies combined with a few physiologic tests can give a presumptive identification to the genus or species level. A schematic flowchart for the tentative identification of medically important aerobic actinomycetes and related genera is given in Fig. 6.2-1 .
Chapter 6.3 : Appendixes
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Actinomadura madurae—causative agent of mycetoma (rare in the United States but more common in tropical and subtropical countries) and nonmycetomic infections (very rare, but some reports of postoperative wound infections, pneumonia, and bacteremia)
7 Mycobacteriology and Antimycobacterial Susceptibility Testing
Chapter 7.1 : General Procedures
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All specimens and cultures handled in the mycobacteriology laboratory must be processed under an appropriate biological safety cabinet (BSC). Personal protective clothing (gowns, gloves, and respirators) is required for processing, smear preparation, and culturing for mycobacteria. Centrifuges must be equipped with aerosol-free safety canisters. Refer to CDC publications for detailed safety recommendations ( 1 , 2 , 4 ).
Chapter 7.2 : Acid-Fast Stains
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Mycobacteria are difficult to stain due to the presence of large amounts of lipid (mycolic acid) in the cell wall; hence, the use of the traditional Gram stain is of little to no value since the dyes do not usually permeate the mycobacterial cell walls. Instead, the most widely used methods to determine acid-fastness in a clinical specimen are the carbol fuchsin stains (Kin-youn or Ziehl-Neelsen) and the fluorochrome stain (e.g., auramine O or r auramine-rhodamine). The fluorochrome stains are recommended for the examination of clinical specimens because of their increased sensitivity and speed, since they may be examined at a lower magnification than the carbol fuchsin stain ( 5 , 7 ).
Chapter 7.3 : Solid Media for Isolation
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Both liquid and solid media are recommended for optimal recovery of mycobacteria. The advantage of solid media (tubed or in plates) is that they enable detection of mixed cultures and contaminants. Egg-based and agar-based media may be used. The main advantage of an egg-based medium is that it supports the growth of most mycobacteria and permits niacin testing. However, contamination occurs more easily involving the total surface of the medium. The main advantages of agar-based media are less contamination and earlier and easier visibility of colonial morphology. Colonial morphology aids in the identification of mycobacteria. Use of both nonselective and selective media is needed for isolation, the latter containing one or more antimicrobial agents to prevent overgrowth by contaminating bacteria or fungi ( 2 ).
Chapter 7.4 : Liquid Media for Isolation
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In the BACTEC 460TB radiometric system (Becton Dickinson Diagnostic Systems, Sparks, Md.), growth medium for culturing mycobacteria is supplemented with a substrate labeled with radioactive carbon (14C). This substrate is utilized by mycobacteria, and during metabolism, carbon dioxide (14C2) is produced from the substrate. The 14C2 is detected quantitatively by counting the radioactivity with a BACTEC 460 instrument. The rate and amount of 14C2 produced are directly proportional to the rate and amount of growth occurring in the medium. This principle is applied for isolation of mycobacteria from clinical specimens, differentiation of the Mycobacterium tuberculosis complex from other mycobacteria, and antimicrobial susceptibility testing.
Chapter 7.5 : Septi-Chek AFB Biphasic Medium
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The BD BBL Septi-Chek AFB mycobacterial culture system (Becton Dickinson Microbiology Systems, Cockeysville, Md.) is a biphasic medium with a self-contained CO2 environment. It consists of Middlebrook 7H9 broth and a slide of Middlebrook 7H11, modified egg, and chocolate media. The system offers sensitivity comparable to those of conventional agar and broth methods ( 1 , 4 ). The inclusion of CHOC in this system is advantageous because it detects contaminants and when incubated at 30°C, Mycobacterium haemophilum can be detected.
Chapter 7.6 : Procedures for Identification from Culture
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Whenever possible, mycobacteria should be identified to the species level. A level laboratory must routinely process and culture at least 20 colonial specimens per week in order to ensure proficiency in identifying Mycobacterium tuberculosis. However, this low workload necessitates referring nontuberculosis mycobacteria to a level III laboratory for identification ( 3 ). In addition to colonial morphology ( 5 ) and acid-fastness, the identification of mycobacteria is largely based on rapid DNA probes (AccuProbe) and conventional methods. The conventional biochemicals used to identify mycobacteria are discussed in this procedure.
Chapter 7.7 : Susceptibility Tests by Modified Agar Proportion
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Drug susceptibility testing should be performedon all initial Mycobacterium tuberculosis complex isolates and in relapse or retreatment cases and when primary drug resistance is suspected. The modified agar proportion method is used to determine the susceptibility of slow-growing mycobacteria (e.g., M. tuberculosis complex, Mycobacterium kansasii), either directly from decontaminated acid-fast smear-positive sputum or indirectly from a pure culture. The agar medium is contained in quadrant plates; one is the control without antimicrobial agents and the other quadrants contain antimicrobial agents. The primary drugs, i.e., isoniazid (INH),ethambutol, and rifampin, are commonly selected for testing; however, pyrazinamide cannot be tested by this method ( 3 , 4 ).
Chapter 7.8 : Susceptibility Tests by Modified Agar Proportion
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The BACTEC 460TB radiometric antimicrobial susceptibility test (Becton Dickinson Diagnostic Systems, Sparks, Md.) is a rapid method based on the principle used for primary isolation of mycobacteria. A 14C-labeled substrate is incorporated in BACTEC 12B medium. When mycobacteria grow in the medium, 14CO2 is produced during metabolism, which is measured as the growth index (GI). If a test drug to which mycobacteria are susceptible is added to this medium, the growth is inhibited, resulting in diminishing GIs as measured by the BACTEC 460 instrument. Radiometric antimicrobial susceptibility testing of Mycobacterium tuberculosis has been well documented and widely used ( 2 , 3 , 6 , 7 ). Results show good agreement with the agar proportion method and are available within 4 to 6 days, compared with 3 weeks with the conventional method.
8 Mycology and Antifungal Susceptibility Testing
Chapter 8.1 : Introduction and General Considerations
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Fungi are significant, sometimes overlooked, human pathogens. Infections range in severity from merely cosmetic to life threatening. Over the last decade, in particular, the spectrum of agents responsible for infection has altered in response to changes in the susceptible population, notably, increases in immunocompromised subjects and greater use of antifungal agents. Fortunately, awareness of the role of opportunistic fungi in disease is growing, but with that awareness the clinical laboratory must be prepared to identify a range of fungal species. In addition, new antifungal agents less toxic than amphotericin B and variously administered have increased the requirement to identify potential pathogens to the species level. (It is no longer acceptable in most situations to report a yeast that does not produce germ tubes as “yeast, not Candida albicans.”) While amphotericin B is effective against many invasive fungal pathogens, newer drugs with a narrower spectrum of efficacy are available (e.g., the echinocandins). Resistance to these drugs is sometimes a species characteristic and may also be isolate dependent for other species. This section brings together the contributions of well-respected mycologists, whose expertise should help laboratories become more adept at identifying fungi, recognizing the significance of an isolated organism, and performing susceptibility testing.
Chapter 8.2 : Specimen Selection, Collection, and Transport
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Suitable specimen selection, proper specimen collection, and rapid specimen transport must be performed to ensure the successful isolation of the etiologic agent of a fungal infection. To establish or confirm the diagnosis of a suspected fungal infection, it is essential for the clinician to provide the laboratory with adequate specimens for evaluation. Also, it is essential for the laboratory to have guidelines for the clinician regarding minimum specimen volumes and appropriate specimen transport (e.g., a laboratory manual or web page). The microbiology laboratory should be notified if an unusual pathogen or an organism that can be a significant laboratory hazard is suspected, as some require special handling or special stains. Examples of unusual fungal and bacterial pathogens, respectively, include Malassezia spp. (some species require the addition of olive oil to culture media) and Nocardia spp. (which are more easily detected on a modified acid-fast stain [see section 6 of this handbook]). Examples of potential laboratory hazards include Coccidioides immitis and Histoplasma capsulatum. Additionally, the microbiology laboratory should be contacted prior to certain procedures, as some specimens for fungal culture may require bedside inoculation onto appropriate culture media (e.g., corneal scrapings).
Chapter 8.3 : Specimen Examination
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The microscopic examination of clinical specimens for the presence of fungi plays an important part in the laboratory diagnosis of most mycoses. Microscopic examination may provide a rapid indication of the cause of an infection, allowing the prompt initiation of appropriate antifungal therapy. It is also important to establish whether the fungus is present in the specimen prior to culture, as some organisms may also occur as laboratory contaminants. Furthermore, the results of microscopic examination may influence the choice of culture media.
Chapter 8.4 : Processing Specimens for Fungal Culture
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When a specimen is suspected to contain a fungal etiologic agent, it should be processed for fungal culture, regardless of direct microscopic findings. Recovery of fungal pathogens in culture provides definitive diagnosis of mycotic disease, identifies the etiologic agent of infection, and allows evaluation of in vitro susceptibility to antifungal agents. In the event that there is insufficient material for both microscopy and culture, all of the specimen should be used for culture, since this is the more sensitive procedure for detection of fungi. Methods of specimen processing and culture are designed to retain the viability of the fungus and to obtain the maximum yield of organisms from clinical specimens. The choice of media for the isolation of fungi from clinical material is based primarily on the most likely species to be found in a particular site or under a recognized clinical condition. Selective media are included when other microorganisms, particularly bacteria, might also be present in the specimen. Specimens should be processed as soon as possible after receipt. Some specimens may require pretreatment prior to culture.
Chapter 8.5 : Examination and Evaluation of Primary Cultures
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Primary plates are read daily for the first week, every other day for the second week, and twice weekly for the remaining 2 weeks. The use of 4 weeks of incubation has been challenged by some groups because few new positive cultures develop in the fourth week. I have noted that clinically significant positive cultures are sometimes seen in the fourth week, suggesting the need to retain this incubation period. In areas of endemicity of systemic dimorphic pathogens, incubation for 5 weeks should be considered, as occasional isolates of Histoplasma capsulatum and Blastomycesdermatitidis may require that much time to form evident colonies. In cases of eumycetoma, the etiologic agent may not be evident on culture until the fifth or sixth week. When growth appears, differentiate between yeast and filamentous forms (moulds) that may require microscopic examination. Use wet mounts or stain with lactophenol cotton blue (LPCB) (seeitem V.B below). If the isolate suggests an actinomycete, examine with Gram stain and with a modified acid-fast stain. (Procedures for the identification of aerobic actinomycetes are given in procedure 6.1, items V.D.1 and V.D.2.)
Chapter 8.6 : Presumptive Identification Tests for Yeasts Isolated on Primary Culture
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All of the tests described in this procedure ( Table 8.6–1 ) are considered presumptive because they do not test a characteristic of a species that is unique to that species. Some of the tests do have high specificity values, which would make the test sufficient for the purpose of medical management of some clinical situations (e.g., intertrigenous candidiasis due to Candida albicans) but insufficient for others (e.g., fungemia due to C. albicans).Presumptive tests also are generally restricted in the range of species they identify. The results of two different physiological tests with high specificity for a particular species may be appropriate for identifying the species presumptively. However, there are instances when two different species elicit identical positive reactions in both tests. Mycologists and clinical microbiologists must be aware of these obfuscations. For example, C. albicans, by far the most frequently encountered Candida sp. in the clinical setting, and Candida dubliniensis are both germ tube positive and positive for the enzymes β-galactosaminidase and l-proline aminopeptidase ( 3 ).
Chapter 8.7 : Identification of Moulds on Primary Culture
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A pure culture is a prerequisite for the identification of a fungus. Fungi may be polymorphic, and any visible structures present must be demonstrated to be produced by one fungus, not several. Bacteria may also interfere with the development of key characteristics.
Chapter 8.8 : Full Identification of Yeasts
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In an era of increasing resistance of yeast species to antifungal agents and a widening range of species capable of causing diseases previously the domain of Candida albicans, there is almost no situation in which identification to species level is not warranted. This is especially true given the growth in the number of immune compromised patients in our society, which has provided more opportunities for yeast infections to occur and to complicate and prolong the recovery period. Molecular methods to identify yeasts directly in specimens and after growth in culture are under development. Since the last edition of this handbook, peptide nucleic acid fluorescence in situ hybridization (PNAFISH) technology (AdvanDx, Woburn, MA) has been developed, which allows identification of several yeasts directly from blood cultures. However, no commercially available method which identifies a broad range of species has been introduced. In this procedure, methods which take advantage of physiological characteristics of various yeast species are presented. Molecular methods are discussed in general terms.
Chapter 8.9 : Mould Identification
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“Mould” is an informal term signifying a fungus growing mostly or entirely in the form of diffuse filaments and usually producing an asexual reproductive state or a sexual state that is not a large, complex fruiting body. Most such fungi are recognized as asexual states or asexual species (the former have known sexual states, while the latter do not) related to various fungi in the phyla Ascomycota and Basidiomycota. In addition, members of the phylum Zygomycota generally grow as moulds.
Chapter 8.10 : Antifungal Susceptibility Testing
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Until recently, antifungal susceptibility testing (AFST) has lagged behind its antibacterial counterpart. A number of important achievements helped propel the antifungal susceptibility field. These achievements include publication of the approved reference method for broth dilution AFST of yeast, document M27-A3 of the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards [NCCLS]) ( 1 , 2 ); publication of interpretive breakpoints using this method ( 10 ); the recommended use of AFST during selection of therapy of patients with candidemia and hematogenously disseminated candidiasis as outlined by the published guidelines for the treatment of candidiasis ( 6 ); publication of a reference method for broth dilution AFST of conidium-forming filamentous fungi, CLSI document M38-A2 ( 3 ); and recent development and publication of AFST methods for dermatophytes ( 3 ). The procedures described below are based on CLSI documents M27-A3, M27-S3, and M38-A2.
9 Parasitology
Chapter 9.1 : Introduction
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If necessary, maintenance procedures can be performed and documented more often than the minimal recommendations presented in this section.
Chapter 9.2 : Collection and Preservation of Fecal Specimens
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One of the most important steps in the diagnosis of intestinal parasites is the proper collection of specimens ( 1 – 4 ). Improperly collected specimens can result in inaccurate results. Fresh specimens are mandatory for the recovery of motile trophozoites. However, unless strict collection and delivery times are adhered to, the specimen may have little value for diagnostic testing.
Chapter 9.3 : Macroscopic and Microscopic examination of Fecal Specimens
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The age of fresh fecal specimens is an important factor in the diagnosis of parasitic infections ( 1 – 4 ). The date and time of passage must be provided for each specimen submitted to the laboratory. The physical characteristics of a fresh fecal specimen may aid in determining what types of organisms may be present ( 1 – 4 ). Fecal specimens are described as formed, semiformed, soft, loose, or watery. Loose or watery specimens may contain trophozoites, whereas formed or semiformed specimens are more likely to contain cyst stages. Helminth eggs or larvae may be found in any type of specimen but are more difficult to find in liquid specimens because of the dilution factor. One can also see if blood and/or mucus is present, although if present, neither one necessarily indicates a parasitic infection. When the fresh specimen is examined visually in the collection container, adult pinworms (Enterobius vermicularis) and tapeworm proglottids may also be seen.
Chapter 9.4 : Special Stains for Coccidia and Microsporidia
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Cryptosporidium and Cystoisospora (Isospora) species have been recognized as causes of severe diarrhea in immunocompromised hosts, but they can also cause diarrhea in immunocompetent hosts. Oocysts in clinical specimens may be difficult to detect without special staining. Cyclospora cayetanensis has also been reported to be acid fast. Modified acid-fast stains are recommended for demonstrating these organisms. Unlike the Ziehl-Neelsen modified acid-fast stain, the modified Kinyoun acid-fast stain does not require heating the reagents used for staining and uses a mild decolorizer ( 1 – 3 ).
Chapter 9.5 : Additional Techniques for Stool Examination
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Strongyloides stercoralis larvae are usually the only larvae found in stool specimens. Depending on bowel transit time and the condition of the patient, rhabditiform and, rarely, filariform larvae may be present. If there is delay in examination of the stool, then embryonated ova and larvae of hookworm may be present. Culture of feces for larvae is useful to (i) reveal the presence of larvae when they are too scanty to be detected by concentration methods, (ii) distinguish whether the infection is due to Strongyloides or hookworm on the basis of rhabditiform larval morphology by allowing hookworm eggs to hatch and release first-stage larvae, and (iii) allow development of larvae into the filariform stage for further differentiation.
Chapter 9.6 : Other Specimens from the Intestinal Tract and the Urogenital System
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The clear-cellulose-tape preparation is the most widely used procedure for the detection of human pinworm infections ( 1 , 2 , 4 ). Adult Enterobius vermicularis worms inhabit the large intestine and rectum; however, the eggs are not normally found in fecal material. The adult female migrates out the anal opening and deposits the eggs on the perianal skin, usually during the night. The eggs, and occasionally the adult female worms, stick to the sticky surface of the cellulose tape. These cellulose tape preparations are submitted to the laboratory, where they are examined under the microscope. Commercial collection systems are also available.
Chapter 9.7 : Sputum, Aspirates, and Biopsy Material
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No descriptions available.
Chapter 9.8 : Detection of Blood Parasites
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During some stages in their life cycle, Plasmodium spp. (malaria), Babesia spp., Trypanosoma spp., Leishmania donovani, and the filariae are detectable in human blood. Plasmodium and Babesia species are found within the RBCs; trypanosomes and microfilariae, the larval stage of filariae, are found outside the RBCs; and Leishmania amastigotes are occasionally found within monocytes. Trypanosomes and microfilariae, which frequently are present in low numbers, exhibit motility in freshly collected blood films, and this can aid in their detection. However, species identifications of all blood parasites are usually made from either or both of two types of stained blood films: a thin film and a thick film. These films can be made from whole or anticoagulated blood or from the sediment of a variety of procedures designed to concentrate trypanosomes and microfilariae in the blood. Although the films are clearest when stained with Giemsa stain, many infections are detected and diagnosed by using Wright's stain. Delafield's hematoxylin is used to enhance the morphological features of microfilariae.
Chapter 9.9 : Culture
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Entamoeba histolytica, the agent of intestinal and hepatic amebiasis, can be cultivated in conjunction with the bacteria voided in feces by the infected patient. Although cultures for E. histolytica are not routinely offered by most clinical laboratories, this approach may be helpful when routine procedures have failed to provide a diagnosis. Polyxenic cultured organisms can also be used to produce intestinal and hepatic amebiasis in susceptible experimental hosts such as hamsters, guinea pigs, and rats. Axenic cultivation of organisms is invaluable for the following: (i) to study the biochemistry, physiology, and metabolism of the organisms in order to establish nutritional requirements of the parasites; (ii) to produce antigens of and monoclonal and polyclonal antibodies against E. histolytica for serological diagnosis as well as other immunologic studies; (iii) to differentiate pathogenic from nonpathogenic strains by using isoenzyme electrophoresis, monoclonal antibody, and/or DNA probes; (iv) to screen drugs in vitro to identify isolates susceptible and resistant to particular drugs so that advances in chemotherapy can be evaluated; (v) to infect experimental animals to produce the disease so that pathological processes can be understood; and (vi) to understand the organization of the parasite at the ultrastructural level.
Chapter 9.10 : Appendixes
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Laboratorian must differentiate extraneous materials present in specimen from actual parasites.
VOLUME 3
Viruses and Chlamydiae
Chapter 10.1 : Laboratory Diagnosis of Viral Infections: Introduction
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The virology laboratory uses several diagnostic modalities, including culture, antigen and nucleic acid detection assays, cytohistopathology, and serologic methods, to aid the physician in the diagnosis of viral infections. The method of choice is influenced by several variables, including the nature of the suspected virus, the availability of test reagents, and the intended purpose of the assay (e.g., detecting active infection, assessing response to therapy). Since no single test modality can satisfy all needs, the laboratory scientist must carefully assess factors such as the patient population and setting as well as the needs and resources of the facility. Furthermore, knowledge of the natural history and pathogenesis of viral infections is essential for the optimal implementation of assays and the interpretation of results.
Chapter 10.2 : Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures
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Viruses are obligate intracellular parasites requiring metabolically active cells to support their replication. While many viruses of diagnostic interest can be cultured in readily available monolayer cell cultures ( Table 10.2-1 ), there are several agents that can be isolated only using specialized systems ( Table 10.2-2 ) such as organ culture (e.g., coronaviruses), leukocyte culture (e.g., the human immunodeficiency viruses and Epstein-Barr virus), or animals (e.g., rabies virus, several coxsackie A viruses, and arboviruses). In addition, there are several important viral agents for which an in vitro system has not been identified (e.g., hepatitis B and C viruses, human papillomaviruses, Norwalk virus, and parvovirus B19).
Chapter 10.3 : Cell Culture Techniques: Serial Propagation and Maintenance of Monolayer Cell Cultures
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Monolayer cell cultures are most frequently used in diagnostic virology. They are prepared by treating the tissue or subculture with proteolytic enzymes and/or chelating agents to dissociate the cells and then seeding the cell suspension in a culture vessel. The cells adhere, divide, and form a layer. Adherence to the vessel surface is important for the subsequent survival and growth of the cells.
Chapter 10.4 : Specimen Collection and Processing
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Successful viral culture requires careful attention to the selection, collection, transport, and assessment of specimens. The information contained in this procedure is essential not only to those laboratories performing viral cultures on site but also to those outsourcing specimens. The laboratory must provide written guidelines to collection sites (nursing station, clinic, physician's office, and emergency room) detailing cultures that are available and instructions for submitting specimens. Instructions should include laboratory hours of operation and contact person(s); instructions for specimen collection, labeling, storage, and transport; source and storage conditions for transport media and containers; information required for adequate testing; turnaround time; reporting procedures and values; and testing limitations.
Chapter 10.5 : Viral Culture: Isolation of Viruses in Cell Cultures
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Viral culture laboratories generally use a combination of tube and shell vial cultures. A number of variables can influence the sensitivity of viral cultures, including cell culture type, age and confluence of the monolayer, the number of tubes or vials inoculated, the inoculation and incubation conditions, and the method and reagent used for isolate detection or identification.
Chapter 10.6 : Isolation of Chlamydia spp. in Cell Culture
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Chlamydiae are obligate intracellular bacteria that contain RNA and DNA, have a cell wall resembling those of gram-negative bacteria, and multiply by binary fission in a manner distinct from those of other bacteria. The 300- to 400-nm spherical elementary body (EB) is the infectious form of the organism. Following cellular infection, the EB reorganizes into a larger, metabolically active reticulate body (RB), which divides repeatedly by binary fission for 24 to 48 h and eventually develops into the characteristic intracytoplasmic inclusion. Human infections associated with the genus Chlamydia are summarized in Table 10.6-1 . Despite the introduction of numerous nonculture assays, including amplified assays, culture remains an important assay for the detection of Chlamydia trachomatis infections and, because of its specificity, is recommended for laboratory testing in cases of sexual abuse and medicolegal situations.
Chapter 10.7 : Direct Detection of Viruses and Chlamydia in Clinical Samples
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Commercially available assays for diagnostic testing are available for several viral agents and for Chlamydia trachomatis ( Table 10.7-1 ) and are discussed in section 12.
Immunology
Chapter 11.1 : Immunology Introduction
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Advances in the clinical immunology laboratory have continued at a rapid pace since the previous edition of CMPH. The goal of this section is to highlight procedures for major assays that are called upon in the clinical microbiology and immunology laboratories and to assist those laboratory directors to conduct the highest quality of laboratory testing. In this section it is impossible to cover all aspects of immunology laboratory testing, as was noted in the previous edition. We therefore again provide Table 11.1.2 from Essential Procedures for Clinical Microbiology ( 2 ), which provides the reader with generic descriptions of assays used in the clinical immunology laboratory. Table 11.1.3 is also provided again as a summary of those assays that are emerging from research laboratories; these may find their way into the clinical immunology laboratory with time.
Chapter 11.2 : Serologic Diagnosis of Group A Streptococcal Infections
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A number of extracellular antigenic products have been identified in cultures of group A streptococci, primarily enzymatic proteins. These include streptolysin O, streptokinase, hyaluronidase, deoxyribonucleases (DNases A, B, C, and D), and adenine dinucleotide glycohydrolase.
Chapter 11.3 : Detection of Legionella Antigen by Direct Immunofluorescence
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Legionnaires' disease is a type of bacterial pneumonia caused by Legionella spp. ( 2 , 14 ). It is estimated that about 1 to 4% of adults with pneumonia requiring hospitalization have Legionnaires' disease and that about 20,000 to 100,000 adults with community-acquired pneumonia have this disease in the United States each year. About 5 to 20% of patients with Legionnaires' disease die of the disease, with major dependence on promptness of specific antibiotic therapy and underlying health of the patient. Legionnaires' disease occurs worldwide, in both epidemic and sporadic form, with sporadic cases being far more common. The disease is more prevalent in some geographic regions than others, for unclear reasons. The primary host risk factors for the disease include immunosuppression, cigarette smoking, and travel. Legionella pneumophila is the cause of more than 90% of cases of community-acquired Legionnaires' disease, with L. pneumophila serogroup 1 being by far the most common causative agent of the disease. Immunosuppressed patients, and those with nosocomial pneumonia, may have infections caused by other L. pneumophila serogroups and other Legionella spp., in particular L. micdadei, L. longbeachae, L. bozemanae, and L. dumoffii. Distribution of the common serogroups and species causing infection may be quite different in various geographic regions. Legionella bacteria are commonly found in the aqueous environment, including tap water and sometimes even distilled water. The preponderance of L. pneumophila as the cause of Legionnaires' disease has resulted in the development of reagents optimized for detection of this species; detection of other Legionella species can be problematic, for reasons of both test sensitivity and specificity. Rarely, organs other than the lungs and pleural space may be infected by the bacterium, causing such diseases as prosthetic heart valve endocarditis, assorted soft tissue abscesses, and systemic infection.
Chapter 11.4 : Urinary Antigen Detection for Legionella spp.
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Patients with Legionnaires' disease excrete soluble serogroup-specific Legionella antigen into their urine. Urinary antigen tests to detect Legionnaires' disease were developed by several groups soon after the 1976 Philadelphia, PA, outbreak of Legionnaires' disease ( 3 , 37 ) and then further refined by Kohler et al. and other groups ( 5 , 16 , 27 – 30 , 34 – 36 ). The antigen being detected has never been extensively purified, but it is known that it is resistant to boiling, is trypsin resistant, has a molecular mass of approximately 10 kDa, and is most likely a lipopolysaccharide ( 29 , 39 ).
Chapter 11.5 : Laboratory Diagnosis of Syphilis
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Syphilis is a sexually transmitted disease that is caused by the organism Treponema pallidum subsp. pallidum. The disease goes through several stages if untreated ( 2 , 15 ). The primary chancre occurs at the site of inoculation approximately 3 to 4 weeks (range, 10 to 90 days) after the initial exposure. Treponemes may be visualized in lesion exudates using either dark-field microscopy or direct fluorescent antibody for T. pallidum (DFA-TP). About 7 to 10 days after the chancre appears, antibodies to T. pallidum are detectable using the routine serologic tests for syphilis. The chancre spontaneously heals after 1 to 4 weeks. The symptoms of secondary syphilis appear about 6 weeks later (range, 2 weeks to 6 months). All serologic tests are generally reactive during secondary syphilis. The most common symptoms are a generalized or localized maculopapular rash that occurs on the palms of the hands and soles of the feet (palmar plantar rash) or on the trunk of the body, mucosal membrane lesions, generalized lymphadenopathy, and condylomata lata. These symptoms will resolve without treatment. The patient then enters a period of latency when there are no symptoms. In about 20 to 25% of individuals, secondary symptoms may reoccur during the early part of this latent period. In early latency (<1 year) the results for the serologic tests for syphilis are reactive. As patients progress into late latency, the nontreponemal tests may become nonreactive, but the treponemal tests will remain reactive. About 65% of persons with untreated syphilis will remain in this stage for life ( 15 ). In the remaining 35%, late manifestations of syphilis will occur.
Chapter 11.6 : Detection of Borrelia burgdorferi Antibodies
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Lyme disease, or Lyme borreliosis (LB), is a multisystem disease caused by Borrelia burgdorferi and transmitted through the bite of infected Ixodes scapularis ticks ( 57 ). It is currently the most frequent vector- borne infectious disease in the United States, with the highest incidence in the northeastern and midwestern states ( 18 ). LB is also prevalent in Europe, where evidence of the disease existed in the early 1900s and where it is transmitted by Ixodes ricinus complex.
Chapter 11.7 : Serodiagnosis of Rickettsial Infections
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Rickettsial diseases in humans result from infection with bacteria of the families Rickettsiaceae and Anaplasmataceae. The family Rickettsiaceae includes the genera Rickettsia and Orientia, both of which infect endothelial cells and thereby cause vasculitis. Rickettsia spp. share lipopolysaccharides and outer membrane protein B (OmpB); however, spotted fever group (SFG) rickettsiae also possess outer membrane protein A (OmpA), whereas typhus group (TG) rickettsiae do not. Since serologic cross-reactivity between groups is common but antibody titers are generally higher within the specific group, distinction between SFG and TG rickettsiae requires paired testing and is not always possible. The Anaplasmataceae family includes Ehrlichia chaffeensis, which infects monocytes and mononuclear phagocytes, and Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, Ehrlichia equi, and the HGA agent) and Ehrlichia ewingii, which infect granulocytes. Serologic cross-reactions occur often (3 to 30%) with Ehrlichia and Anaplasma, as they do with Rickettsia, and differentiation requires comparison of specific antibody titers.
Chapter 11.8 : Immunoassay Detection of Shiga Toxin-Producing Escherichia coli
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Shiga toxin-producing Escherichia coli (STEC) organisms are responsible for a broad range of gastrointestinal illnesses, from mild watery diarrhea to severe hemorrhagic colitis associated with hemolyticuremic syndrome (HUS) ( 1 , 5 , 21 , 30 , 34 , 36 ). These organisms cause disease primarily through elaboration of one or more Shiga toxins (Stx1, Stx1 variants, Stx2, and Stx2 variants) encoded by genes carried on lambda bacteriophages ( 10 , 14 , 34 , 36 ). Research supports the fact that hemorrhagic colitis and HUS likely result from the action of these toxins on vascular endothelium since Stx targets digalactosyl receptors on microvascular endothelial cells in the gut, kidneys, and brain ( 30 , 34 , 36 ). E. coli organisms that produce Stx2 alone have been found to be more virulent than isolates that have Stx1 alone or that produce Stx1 and Stx2 in combination ( 34 ). Enhanced virulence may also be related to host factors and genetic differences among strains. Some investigators have reported differences in the frequency and distribution of Stx genes in the type of clinical disease reported ( 24 , 30 ). Hence, genotyping may be important not only in characterizing particular outbreaks but also in predicting disease severity and treatment ( 18 , 24 , 30 ). Among other organism virulence factors important for pathogenesis are an adhesin, intimin, encoded by an eae gene, and enterohemorrhagic E. coli (EHEC) hemolysin (ehxA gene), a potent cytolysin ( 34 , 36 ).
Chapter 11.9 : Serodiagnosis of Helicobacter pylori
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In 1983, Marshall and Warren proposed the possible association of Helicobacter pylori with peptic ulcer disease and gastric cancer ( 17 ). In February 1994, the NIH Consensus Development Conference concluded that H. pylori infection is the major cause of peptic ulcer disease and that all patients with documented peptic ulcer disease associated with H. pylori infection should receive antimicrobial therapy ( 20 ).
Chapter 11.10 : Total Viable Cell Counting Procedure
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Trypan blue allows rapid observation of nonviable cells present in a peripheral blood mononuclear cell suspension (PBMCS). A count of viable cells present in a PBMCS allows the suspension to be adjusted accordingly for further use in assays.
Chapter 11.11 : Peripheral Blood Mononuclear Cell Cryopreservation Method
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To be able to cryopreserve peripheral blood mononuclear cells (PBMCs) for utilization in future testing
Chapter 11.12 : Lymphocyte Proliferation Assay
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Measuring the proliferative capability of lymphocytes is important in the evaluation of an individual's immune system. This can be achieved in an assay which cultures lymphocytes with either a mitogen or antigen that can stimulate a proliferative response and then labels the cells with a radioactive marker to allow for measurement of response ( 1 ).
Chapter 11.13 : Natural Killer Cell Assays
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Natural killer (NK) cells are another group of cytolytic lymphocytes, distinct from B lymphocytes and T lymphocytes, that participate in both innate immunity and adaptive immunity. NK cells are lymphocytes that lack B-cell receptors and T-cell receptors. They are designed to kill certain mutant cells and virus-infected cells. Normally present as a small subpopulation of circulating lymphocytes, NK cells morphologically appear as so-called “large granular lymphocytes.” Like cytotoxic T cells, they attack and kill tumor cells and protect against a wide variety of infectious microbes. They are “natural” killers because they do not need additional stimulation or to recognize a specific antigen in order to attack and kill. NK cells appear to play a role in a variety of human diseases. Compromised or absent natural immunity, as measured in vitro by decreased NK activity and/or depressed absolute numbers of circulating NK cells, has been linked to the development and progression of cancer, chronic and acute viral infections (including AIDS), various immunodeficiencies, and certain autoimmune diseases.
Chapter 11.14 : Quantitation of Human Interleukin 4, Interleukin 6, and Gamma Interferon
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Interleukin 4 (IL-4), IL-6, and gamma interferon (IFN-γ) are cytokines with many cellular effects, including mediation of the immune response to infection. Among other biological activities, IL-4 and IL-6 stimulate B cells and IFN-c has antiviral activities ( 6 ). There is an increasing interest in using cytokine levels to monitor disease progression ( 2 ). There are several commercially prepared kits available to quantify cytokines by ELISA. This procedure is written for use with kits manufactured by Pierce Endogen. If IL-4, IL-6, or IFN-c is present in the clinical specimen, it will bind to an ELISA plate that is precoated with antibody specific for that cytokine. After the patient specimen is incubated with a biotinylated detection antibody, the plate is washed and then streptavidin conjugated to horseradish peroxidase (HRP) is added. After another incubation, tetramethylbenzidine (TMB) substrate is added to the plate. The enzyme-substrate reaction produces a color change that can be measured spectrophotometrically. The unknown values can be determined from a standard curve in which the color intensity is directly proportional to the standard concentration.
Chapter 11.15 : Flow Cytometry Whole-Blood Intracellular-Cytokine Assay Using Phorbol Myristate Acetate, Ionomycin, and Brefeldin A
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Cytokines are soluble proteins produced by T and B lymphocytes, natural killer cells, monocytes, macrophages, and granulocytes. Specifically, cytokines regulate growth, differentiation, and function of a wide variety of cells and mediate normal and pathological responses. Most cytokine bioassays examine cytokines at the cell population level, but they cannot provide information concerning the phenotype of cytokine-producing cells or mechanism of cytokine products. The flow cytometry method described here measures production of cytokines by T-cell subsets using whole blood. Heparinized blood is stimulated with the polyclonal activator phorbol myristate acetate plus ionomycin (induces intracelluar signal cascades for polyclonal leukocyte activation) and the protein transport inhibitor brefeldin A (BFA) (a fungal metabolite that interferes with vesicular transport from the rough endoplasmic reticulum to the Golgi complex) for 4 h at 37°C and 5% CO2. Activated cells are stained with monoclonal antibodies for lymphocyte surface markers, followed by lysing of RBCs. WBCs are fixed and permeabilized simultaneously and stained with monoclonal antibodies to intracellular cytokines. Stained cells are analyzed by flow cytometry.
Chapter 11.16 : Whole-Blood Lymphocyte Immunophenotyping Using Cell Surface Markers by Flow Cytometry
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Whole blood can be stained with fluorochrome-labeled monoclonal antibodies against antigen markers found on the surfaces of lymphocytes. The stained samples are treated with a lysing solution to destroy erythrocytes. The flow cytometer is an instrument capable of rapid, quantitative, multiparameter analysis of heterogeneous cell populations on a cell-by-cell basis (single-cell analysis). During acquisition, the cells travel past the laser beam and scatter the laser light. The stained cells fluoresce. These scatter, and fluorescence signals provide information about the cells' size, internal complexity, and relative fluorescence intensity. The percentage of fluorescent cells is determined for each antibody. In addition, the use of the light scatter measurements from the individual cells along with fluorescence allows identification of the lymphocyte population.
Chapter 11.17 : Neutrophil Function Whole-Blood Flow Cytometric Test for Leukocyte Adhesion Deficiency
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Normal neutrophil function is of great importance in the host defense against bacterial and fungal infections. As neutrophils recognize, adhere to, and phagocytose an invading microbe they generate an oxidative burst, which kills the pathogen. In the leukocyte adhesion deficiency (LAD) states, there are defects in the ability of neutrophils to adhere to both the vascular endothelium and opsonized microorganisms due to the absence or reduced expression of a group of cell surface leukocyte adhesion markers. This group of surface glycoproteins includes LFA-1 (CD11b/CD18), Cr3 (CD11b/CD18), and P150,95 (CD11c/CD18). Patients with this deficiency manifest recurrent infections involving the skin, subcutaneous tissues, middle ear, and oropharyngeal mucosa. In the assay described here, neutrophils are stimulated with phorbol 12 myristate 13 acetate (PMA) and stained with CD11b plus corresponding isotypic monoclonal antibody. Cellular fluorescence is measured on resting and stimulated neutrophils using flow cytometry.
Chapter 11.18 : Flow Cytometric Test for Chronic Granulomatous Disease
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Normal neutrophil function is of great importance in the host defense against bacterial and fungal infections. As neutrophils recognize, adhere to, and phagocytose an invading organism, they generate an oxidative burst which results in the reduction of molecular oxygen to superoxide. The superoxide produced is rapidly converted to hydrogen peroxide (H2O2, which kills the pathogen). In chronic granulomatous disease (CGD), microbial killing is defective because neutrophils from patients with CGD lack a respiratory burst. The assay described here uses dihydrorhodamine-123 (DHR-123) and phorbol 12 myristate 13 acetate (PMA) to measure oxidative burst activity, which is easily converted to hydrogen peroxide. DHR, a nonfluorescent compound, reacts with hydrogen peroxide and is oxidized to rhodamine-123, a green fluorescent compound. Cellular fluorescence is measured using flow cytometry.
Chapter 11.19 : Gamma Interferon Enzyme-Linked Immunospot Assay
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The enzyme-linked immunospot (ELISPOT) assay was described more than 13 years ago for the detection of specific immune cells at the single-cell level. The utility of the gamma interferon (IFN-γ) ELISPOT assay in detecting antigen-specific T cells was initially demonstrated in models of autoimmune and infectious diseases. Optimization of the assay through the introduction of specifically designed antibodies, 96-well plates, substrate kits, and other modifications has broadened the potential uses for the IFN-γ ELISPOT assay. Today, it is being used for a wide range of applications, including monitoring responses in patients with cancer undergoing immunotherapeutic treatment and monitoring specific immune response patterns in patients with infectious, neoplastic, or autoimmune diseases. Additionally, it has been an important tool in the identification of immunodominant epitopes in human immunodeficiency virus and Mycobacterium tuberculosis infections, as well as in the development of specific vaccine strategies ( 1 ).
Molecular Diagnostics
Chapter 12.1 : Introduction
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Molecular techniques have been used successfully to aid in the detection and identification of pathogens and for the treatment of many infectious diseases ( 1 , 4 , 7 , 9 , 10 , 12 ). Some of the earliest diagnostic applications included direct nucleic acid probes for identification of bacterial or fungal isolates and for direct detection of microorganisms in patient samples. This was followed by the introduction of nucleic acid amplification techniques (NAATs) for which utility in clinical testing was originally complicated by the complexity of the testing processes. These processes included many manual steps, such as nucleic acid extraction, amplification, and detection, and methods were fraught with the potential for false-positive results due to a combination of contamination risks plus the exquisite sensitivity of the testing methods. With the acquisition of expertise and dissemination of information regarding appropriate molecular laboratory procedures, including the production of CLSI guidelines ( 8 ) and the recent inclusion of all molecular infectious disease testing into the CAP microbiology checklist ( 3 ), along with the advent of newer technologies, such as automated extraction and real-time detection, use of molecular techniques in the clinical setting has become more commonplace and is no longer considered to be an esoteric or specialized practice reserved for reference laboratories. This is best exemplified by the fact that molecular testing systems are now available for use in moderate-complexity laboratory settings.
Chapter 12.2 : Molecular Methods for Direct Detection of Microorganisms in Clinical Specimens
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The detection of pathogenic microorganisms directly in clinical specimens by molecular methods has been investigated extensively using a variety of nucleic acid probe hybridization, target amplification, and signal-generating formats. Commercial product development has focused on direct diagnosis of blood-borne and sexually transmitted diseases and respiratory pathogens using solid-and solution-phase hybridization with nonisotopic nucleic acid probes and several different target amplification methods, including conventional PCR, strand displacement amplification, transcription-mediated amplification, and, more recently, real-time PCR. Many laboratories are developing their own “home brew” assays for pathogen detection using real-time PCR for organisms not addressed by commercial kits (Table 12.1-4).
Chapter 12.3 : Molecular Methods for Identification of Cultured Microorganisms
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Bacteria and fungi can be identified by using nucleic acid hybridization techniques. Nucleic acid probes can be used to confirm the identification of culture isolates that have been tested by presumptive identification methods. Alternatively, probes can be used as the primary method for identifying isolated organisms. Culture identification by probe hybridization is not dependent on the ability to detect minute quantities of nucleic acid, and thus sensitivity is not a limiting factor in this application of molecular technology. The advantage of probe-based identification is greatest for slow-growing organisms like the mycobacteria or for organisms for which convenient commercial identification systems are not available. Although the specificity of the available commercial probes is high and they facilitate rapid identification of a number of pathogens (Table 12.1-3), misidentifications do occur and serve to emphasize the need for caution in using any single characteristic in identification of a species. It should also be emphasized that at this point identification by probe hybridization is more expensive than conventional techniques for many organisms.
Chapter 12.4 : Molecular Methods for Epidemiologic Typing of Microorganisms
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The ability to identify specific strains within a species of pathogen is an important aid in the rational development of effective measures to prevent and control nosocomial infections. The efforts of both microbiologists and hospital epidemiologists are facilitated greatly by the availability of the newer molecular epidemiologic typing techniques. The variety of molecular epidemiologic tools available at present is considerable, and based on current experience the methods that appear to be the most practical and useful for both large- and small-scale epidemiologic studies are the DNA-based methods such as pulsed-field gel electrophoresis (Table 12.1-5). Although these methods clearly have limitations, they generally are a significant improvement over the more conventional typing methods, many of which are too cumbersome, insensitive, and time-consuming to be of practical value for epidemiologic evaluations. It is important to understand that no single technique is universally applicable and that the choice for a particular application is related to the species studied, the scope of the question posed, and the convenience of the technique. The techniques of molecular epidemiology are useful in answering real clinical and infection control questions and are not limited to research uses. Examples include distinguishing between relapse and reinfection in an individual patient and in tracking the spread of an individual strain of a bacterium or fungus within the hospital environment ( 1 – 3 ).
Chapter 12.5 : Molecular Methods for Antimicrobial Agent Resistance Determination
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Antimicrobial agent resistance is an increasing problem worldwide, particularly among critically ill hospitalized patients. For this reason, there is a renewed interest in monitoring the development and spread of antimicrobial agent resistance and a recognition of the need for effective interventions to limit the spread of resistance to prolong the therapeutic life of the available antimicrobial agents. Unfortunately, conventional methods to perform antimicrobial agent susceptibility testing may be too slow and insensitive in detecting antimicrobial agent resistance to be of much use clinically. The techniques of molecular biology have been used to characterize resistance at the DNA level and may provide rapid, sensitive, and specific information to the clinician for use in therapeutic decision making ( 1 – 4 ). Genetic material that confers antimicrobial agent resistance may be carried on the bacterial chromosome or on transposons or plasmids and has been detected by probe hybridization or by DNA amplification with PCR (Table 12.1-6). Molecular detection of resistance has potential value for decisions directly related to patient care and is useful for calibration of conventional susceptibility tests and for precise definition of the mechanisms of resistance to selected antimicrobial agents. Molecular techniques have been used to detect genes encoding several different mechanisms of resistance against antimicrobial agents, e.g., β-lactam agents, aminoglycosides, macrolides, and fluoroquinolones, after isolation of a clinical isolate (Table 12.1-6).
Chapter 12.6 : Appendix 12.6-1—Unidirectional Work Flow and Minimization of Contamination
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The extraordinary sensitivity of nucleic acid amplification methods makes them invaluable tools for detecting infectious agents. However, the ability to detect small quantities of nucleic acids also makes these techniques prone to false-positive results due to specimen-to-specimen cross-contamination or carryover contamination from previously amplified products. The risk of contamination can be greatly reduced by maintaining a unidirectional work flow and by strictly adhering to appropriate QC procedures.
Epidemiologic and Infection Control Microbiology
Chapter 13.1 : Introduction
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Clinical microbiologists have long been comfortable with the analytical contributions they make to laboratory diagnosis. However, the total testing process for the microbiologist additionally includes preanalytical and postanalytical issues about which a technologist may feel less secure. Clearly, one major area of contribution unique to the clinical microbiologist is the critical data expertly provided to the infection control team of the institution. A sound infection control program of any health care institution is dependent on its relationship with the microbiology laboratory from which most of their critical data come. While accuracy and timeliness of critical microbiology diagnostic reports contribute to positive patient outcomes, the nature of skilled interpretive judgment on the part of trained technologists marks the clinical microbiologist as a uniquely qualified contributor to the data that drive infection control decisions.
Chapter 13.2 : Laboratory Support for Infection Control: Optimization by Policy and Procedure
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The discipline of infection control had its formal beginnings in the 1970s and was refined in the 1980s. This discipline will continue to evolve, becoming part of the health care providers' overall program of continuous quality improvement. Super-imposed on the evolution of infection control programs has been the rapid evolution of the health care field, introducing new modalities of care that are prone to high rates of nosocomial infection. Fewer patients are hospitalized, but in general, those who are hospitalized are sicker than similar populations in previous years. The numbers of indwelling lines, implanted devices, and complex procedures have greatly increased, as have the numbers of patients compromised by cancer chemotherapy regimens or transplant immunosuppressive therapy. Transfusion practices have evolved to include a variety of processed subsets of blood, and transplantation now includes many solid organs, bone, bone marrow, corneas, and various connective tissues. In addition, special patient populations such as the elderly, neonates, women, or ethnic minorities are being evaluated for their special health care needs that might require unique services. The microbiology laboratory is increasingly being asked to perform cultures that support the needs of these new programs. The involvement of the laboratory must be optimized by appropriate policies and procedures and the appointment of the chief microbiologist to the Infection Control Committee. With early patient release and with multiple hospitals often served by the same microbiology core laboratory, it is imperative that the laboratory develop methods and communication tools to assist in serving the patients' infection control needs. The Institute of Medicine in 2000 addressed the issue of patient safety, and although it did not specifically focus on health care-associated infections, these are clearly critical areas for improvement and monitoring.
Chapter 13.3 : Policies for Environmental Sampling and Culturing for Infection Control
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Sampling of the environment should be performed only to answer a specific question, whether to support a defined hypothesis or to confirm an association generated by an epidemiologic investigation. Culturing for infection control purposes should be systematic, consistent, and part of a written infection control plan that is reviewed at least yearly and is specific to each facility. Arbitrary sampling risks generation of irrelevant or uninterpretable information. Worse, such information may be frankly misleading. In each case, inappropriate culturing can result in wasted effort and resources and may even cause harm to patients or personnel. For example, if arbitrary culturing identifies an irrelevant source of an organism, the epidemiologically important source may go undetected, leading to further unnecessary exposures.
Chapter 13.4 : Outbreak Investigations: Laboratory and Epidemiologic Concepts
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Epidemiologic investigations may be performed in response to a change in the rate of an event or outcome compared with a community or facility's baseline or to evaluate a rate that is considered unacceptable compared with published rates in similar populations ( 20 ). The degree of departure from baseline needed to identify an outbreak is variable, depending on factors such as the population in which it occurs and the organism or agent responsible. A single case of a rare or virulent infection, e.g., West Nile encephalitis, rabies, measles, or cholera, may be enough to herald an outbreak. For more common or endemic organisms, like methicillin-resistant Staphylococcus aureus (MRSA), an outbreak may mean a significant increase above baseline rates of clinical isolates or illness. Thus, specific thresholds for concern vary from case to case. If a particularly fragile population is involved, e.g., low-birth-weight infants in the neonatal intensive care unit or patients in a burn unit, the threshold for investigation may be lower. In addition to the pathogen and the population involved, outbreaks may be defined by a change in prevalence of certain organism characteristics, e.g., antimicrobial susceptibility or toxin production. In each of the above cases, meaningful surveillance data against which to measure a change are a critical component of outbreak investigation ( 12 ).
Chapter 13.5 : Epidemiologic Strain Typing
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The diversification of health care settings (e.g., home health care, nursing homes, assisted living, or outpatient dialysis centers) presents new challenges for the clinical microbiologist, hospital epidemiologist, and infection control professional as they work together to detect and investigate disease trends and outbreaks. A valuable tool in epidemiologic investigations is strain typing. This procedure is a quick guide that offers a simplified list of typing techniques of potential utility to clinical microbiologists. Readers seeking in-depth descriptions or detailed protocols should refer to References and Supplemental Reading.
Chapter 13.6 : Culture of Hospital Water for Legionellaceae
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No descriptions available.
Chapter 13.7 : Culture and Endotoxin Assay of Hemodialysis Fluids
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Patients undergoing hemodialysis are at risk for the development of endotoxin-mediated pyrogenic reactions, gram-negative bacteremia with sepsis, and chronic inflammatory response syndrome. These events may be related to excessive levels of gram-negative bacillary contamination of the water and dialysate used for hemodialysis applications. This cause-and- effect relationship has led to the establishment of microbiological guidelines pertaining to the allowable levels of contamination in both the dialysate bath and dialysis water. These recommendations include frequency of monitoring and a list of methods considered acceptable for the performance of quantitative testing. Endotoxin testing is discussed in the appendixes at the end of this procedure because it is not routinely done in the clinical setting and is performed usually by renal reference laboratories.
Chapter 13.8 : Culture of Peritoneal Dialysis Fluid
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No descriptions available.
Chapter 13.9 : Air Cultures for Fungi
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Air is the medium that transports fungi from sources such as soil, construction disturbances, or moldy hospital environments ( 7 , 14 , 16 ). Although breathing airborne fungi is common and for most individuals results in no effects on health, hospitalized patients with extreme immune suppression are susceptible to infections with those airborne fungi that can grow at body temperatures ( 11 ).
Chapter 13.10 : Microbiological Assay of Environmental and Medical-Device Surfaces
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No descriptions available.
Chapter 13.11 : Surveillance Cultures from Immunocompromised Hosts
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Surveillance cultures from immunocompromised patients provide a service that can directly impact patient care and outcome in this patient population, but a rational, planned approach must be considered in order to optimize resources and interpretive impact. While some studies suggest that surveillance cultures may be of value, other studies suggest that routine surveillance may not be helpful. Differing conclusions from these studies may be due to insufficient data or inconclusive definitions of test conditions rather than from opposing data for similar populations. Selective surveillance, depending on occurrences at a particular time, can be useful in predicting anticipated difficulties in immunocompromised individuals. Surveillance could be part of studies to define the natural history of colonization by microorganisms and infections during pretreatment and after treatment of the immunosuppressed patient.
Chapter 13.12 : Culture of Intravascular Devices
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Intravascular catheters are used to provide continuous vascular access to permit blood sampling; to administer blood products, medications, total parenteral nutrition, and other fluids; and, in the case of pulmonary artery catheters, to permit hemodynamic monitoring of cardiac function. Because these devices penetrate the integument, they put the patient at significant risk for development of device-related infection. The insertion site becomes colonized by bacteria from the patient's own skin or by microorganisms carried on the hands of medical personnel ( 5 ). Organisms can also gain access through the lumen of the catheter following contamination of the hub ( 4 ) or infusion of contaminated fluids. Invading organisms can then colonize the intravascular catheter surfaces in the form of a biofilm and produce local infection and, in a significant number of cases, bacteremia, fungemia, suppurative phlebitis, or septic thrombosis ( 5 ). (See Appendix 13.12-1 for further discussion.)
Chapter 13.13 : Culture of Blood Bank Products
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Transfusion of blood and blood components is usually a safe and effective form of therapy. However, untoward effects can occur. These untoward effects, called “transfusion reactions,” may present immediately or be delayed, and they may or may not be immunologically mediated. A serious complication of transfusion therapy is the transmission of infection by many different kinds of microorganisms, such as viruses (hepatitis) ( 7 ), spirochetes (Borrelia burgdorferi) ( 4 ), and other bacteria, for example, Yersinia spp. This procedure focuses only on culture of suspected bacterial agents in blood and blood products. A list of some bacterial agents implicated in transfusion reactions is shown in Table 13.13-1 .
Chapter 13.14 : Microbiological Assessment of Orthopedic Surgery Sites
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The severe morbidity associated with infection following implantation of orthopedic prosthetic devices or repair of bone fractures at septic trauma sites requires early and accurate microbiological assessment of these sites for successful management. Although no procedural or interpretive standards for culture of these sites have been established, the following is a set of procedures often used by surgical teams to assess microbial load or, along with other laboratory findings, to confirm the presence of an infection. These procedures include (i) enumeration of bacteria in tissue from a contaminated trauma site prior to repair of fracture, (ii) intraoperative culture of a site selected for implantation of a prosthesis, (iii) culture of fluid removed from the joint, and (iv) intraoperative culture of a failed prosthesis site.
Chapter 13.15 : Quantitative Culture of Small-Bowel Contents
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The microbial content of the normal gastrointestinal tract varies with the precise site ( 2 , 5 ). The concentration of bacteria in gastric aspirates from fasting patients is fewer than 103/ml. Duodenal and jejunal organisms are normally present at fewer than 105/ml ( 3 ) and consist of primarily gram-positive bacteria (staphylococci, streptococci, and lactobacilli) and yeasts. Members of the family Enterobacteriaceae are occasionally present in small numbers. Strict anaerobes are notably absent. Postprandially, the microbial content of the proximal small bowel is increased by the introduction of food and oropharyngeal microorganisms. The ileal microbiota more closely resembles that of the colon, with the ileocecal valve incompletely regulating backwash from the colon. In the distal ileum, the bioload consists of 107 to 108 aerobes and anaerobes per ml, whereas in the colon, a significant rise in anaerobes to 1010 to 1011 organisms per ml and of aerobes to 108 organisms per ml is found ( 3 ). (See Appendix 13.15-1 for further discussion.)
Chapter 13.16 : Phenotypic Characterization of Organisms for Epidemiologic Purposes
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Numerous methods exist for characterizing microorganisms beyond the species level. Methods include but are not limited to determining antimicrobial resistance patterns, biotyping, serotyping, and using molecular techniques such as plasmid analysis and restriction enzyme analysis of plasmid or chromosomal DNA. These methods are used when it is necessary to separate organisms more finely for clinical and epidemiologic purposes. In order to have wide application, laboratory techniques for typing organisms in the clinical microbiology laboratory must be easy to perform with readily available reagents and materials. A valid typing technique must be reproducible, standardized, and stable over time. It must be sensitive enough to distinguish epidemiologically related and unrelated strains. No single method of strain subtype delineation has proved to be ideal. Most often, a combination of several systems of characterization is used to evaluate a group of organisms. The fundamental principle in the application of any technique to the typing of organisms is that the entire group must be tested as a batch. All organisms must be run in parallel on the same day by the same laboratory and the same personnel with identical reagents. (See Appendix 13.16-1 on p. 13.16.5.1 for further discussion.)
Chapter 13.17 : Screening for Methicillin-Resistant Staphylococcus aureus
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Screening patients for colonization with methicillin-resistant Staphylococcus aureus (MRSA) is often used during outbreaks to identify and isolate colonized/ infected patients in an attempt to prevent further spread on health care units and within health care facilities. The utility of ongoing surveillance programs and universal admission screening is controversial at present ( 1 , 3 – 5 , 8 , 9 ) but they are used in some jurisdictions. The microbiology laboratory must provide input into screening programs that are implemented and be reimbursed for procedures that are part of these programs. The sensitivity of any screening program depends on the specific body sites sampled, the number of sites sampled, and the methods used for detection. The laboratory costs for screening initiatives are often high, so these initiatives should not be undertaken at the expense of less costly infection control practices.
Chapter 13.18 : Screening for Vancomycin-Resistant Enterococci
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Vancomycin-resistant enterococci (VRE) (also referred to as glycopeptide-resistant enterococci) have emerged as significant nosocomial pathogens. Immunosuppression and indwelling devices are risk factors for invasive infections ( 3 , 6 ). Transmission on hospital wards is the major source of spread of these antibiotic-resistant organisms. Infection rates can be reduced by screening high-risk groups ( 4 ). Patients colonized with VRE are isolated to prevent transmission. The laboratory, in conjunction with the Infection Control Program, should decide which groups of patients to screen and the frequency of screening depending on the local epidemiology, unless regulations state otherwise ( 8 ).
Quality Assurance, Quality Control, Laboratory Records, and Water Quality
Chapter 14.1 : Quality Assessment and Improvement (Quality Assurance)
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QA is a process of monitoring the functional components of a system and correcting defects when unacceptable performance is identified. Quality is characteristically assessed by specifying performance indicators and setting targets (thresholds) for acceptable proficiency. Limits may be set so that action is taken only when the number of deficiencies exceeds a specified threshold, or a limit may be defined as a sentinel event that requires review and action whenever it is encountered. The functional attributes of a QA plan are listed in Table 14.1-1 .
Chapter 14.2 : Quality Control
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QC programs ensure that information generated by a laboratory is accurate, reliable, and reproducible. This is accomplished by assessing the quality of the specimens; monitoring the performance of test procedures, reagents, media, instruments, and personnel; reviewing test results; and documenting the validity of the test methods.
Chapter 14.3 : Laboratory Records
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“If it hasn't been recorded, it hasn't been performed.” This sentence summarizes the absolute requirement for detailed and accurate record retention for all aspects of the clinical microbiology laboratory. Records serve at least four purposes. (i) They document what has transpired without recourse to memory (they provide a paper or electronic audit trail), (ii) they serve as a point of reference for developing the facts regarding any incident, (iii) they assist in the recognition of trends and the resolution of problems, and (iv) they establish the laboratory's credibility. The standards for record keeping and the length of time records must be maintained are established by the agencies presented in Appendix 14.3-1 . Federal guidelines should be considered minimum standards and are superseded by the standards established by the states or other certification agencies.
Chapter 14.4 : Preparation and Quality Control of Laboratory Water
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The objectives of this procedure are to describe methods of preparation and QC testing of water used in microbiology laboratories and to compare water specifications published by the NCCLS ( 4 ), the American Society for Testing and Materials, the Environmental Protection Agency ( 2 ), the American Public Health Association-American Water Works Association-Water Pollution Control Federation ( 3 ), CAP ( 1 ), the American Chemical Society, and the U.S. Pharmacopeial Convention.
Biohazards and Safety
Chapter 15.1 : Introduction
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Laboratory workers are at high risk for occupational exposure to infectious agents. Infections can be acquired from exposure to contaminated blood, tissue, and other material. The greatest risks for clinical microbiologists are associated with the processing of specimens and the manipulating of pathogens isolated from these materials. The actual incidence of laboratory-acquired infections is probably higher than recognized due to subclinical symptoms and poor compliance in reporting.
Chapter 15.2 : Biological Safety and Biohazard Prevention
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No descriptions available.
Chapter 15.3 : Biohazard Containment
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In addition to implementing standard microbiological procedures and practices, management of the biohazards associated with working with pathogens includes physical barriers and administrative controls. Physical barriers include primary safety equipment and secondary facility design. Procedures 15.3.2 through 15.3.5 describe general physical and administrative controls for microbial containment.
Chapter 15.4 : Laboratory Instrumentation and Equipment
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Procedures 15.4.2 through 15.4.6 describe common operational procedures performed in the clinical microbiology laboratory. They are intended to present proper safety practices when working with equipment and handling infectious material.
Chapter 15.5 : Special Pathogens and Employee Safety
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The purpose of the information in this subsection is to help reduce the chances of clinical microbiology laboratory employees' being exposed to microorganisms associated with bioterrorism and with laboratory-acquired infections, including Creutzfeldt-Jakob disease.
Chapter 15.6 : Packing and Shipping Infectious Substances
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The information in this procedure is not intended to be an all-inclusive guide to packing and shipping regulations. The information is a summary of my interpretations of the current (as of 1 January 2009) requirements and regulations issued by the following: the International Civil Aviation Organization (ICAO; a specialized United Nations [UN] agency which promotes the international standardization of essentially all technical aspects of aviation, including the transport of dangerous goods), the International Air Transport Association (IATA; a commercial airline trade association), and the U.S. Department of Transportation (DOT; an agency of the federal government).
Chapter 15.7 : Management of Laboratory Accidents
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Despite improved control measures (engineering controls, safe work practices, and PPE), laboratory workers remain at risk for acquiring laboratory-associated infections. Reported cases of fatal meningococcemia in clinical laboratory workers underscore the potential risks of handling clinical samples ( 2 ). Every laboratory (e.g., anatomic pathology, clinical diagnostic, reference, and research laboratories) should implement a biosafety plan. The essential components of the plan should include written procedures to reduce risks of occupational exposure and mandatory training, health assessment of employees, and record keeping of all exposures and treatments. A risk assessment of the procedures carried out on samples, including the frequency of positive samples, should be determined. The potential risk of disease should also be evaluated along with the availability of postexposure prophylaxis (PEP) and preventive vaccines. The benefits and side effects of PEP and immunization must also be considered.
Chapter 15.8 : Management of Infectious Waste
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For effective and efficient management of infectious wastes, a comprehensive waste management plan is essential in order to ensure the safety of the employees handling the waste, compliance with the various regulatory requirements ( 14 , 17 , 18 , 20 ), meeting the standards of the JCAHO ( 11 ) and the guidelines of other professional groups such as the NCCLS ( 13 ), and implementation of cost-effective strategies for wasste disposal. In developing a comprehensive plan for infectious-waste management, consider the following factors because of the constraints that they impose.
Bioterrorism
Chapter 16.1 : General Introduction to Bioterrorism
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Since the events of 11 September 2001 and the subsequent release of anthrax in October 2001, bioterrorism preparedness has been a major priority for the nation. As an integral member of the “first responder” team in recognizing or suspecting an act of bioterrorism, the clinical laboratory, especially the clinical microbiology laboratory, will serve as a sentinel in the detection, recovery, characterization, and identification of the targeted biological agent(s). In preparation for responding to a biological terrorism event, the clinical microbiologist is encouraged to participate in and apply the guidelines of the Laboratory Response Network (LRN). Members of the laboratory staff should be formally trained and knowledgeable in the following areas: (i) the BSL of their laboratory; (ii) principles of specimen collection, preservation, packaging, labeling, and shipment; (iii) criteria for recognizing or suspecting a potential bioterrorist activity and the institutional chain of communication; (iv) biothreat levels as designated by the LRN; (v) diagnostic testing according to consensus protocols; (vi) timely and accurate testing and reporting; and (vii) the chain of communication linking local, state, and federal agencies. Although it is not anticipated to be a major factor, members of the microbiology staff should have an understanding of the chain-of-custody guidelines being practiced in their institution. It is vital for a laboratory to be familiar with its role in response to a suspected or confirmed bioterrorism event and to develop formal standard operations procedures (SOPs), which describe how the laboratory will function in the event of a biological incident. The SOP should be part of an institution- wide SOP that is a multidisciplinary document comprised of policies from Infection Control, Public Relations, Risk Management, Pharmacy, Security, Medical Staff, and Administration. The primary role of the clinical microbiology laboratory in responding to a bioterrorism event will be no different from its present role: to detect, recover, and characterize or identify the etiological agent(s). Of utmost importance is maintaining awareness that an event may be occurring and raising suspicion that requires further investigation. Secondary roles include maintaining an active surveillance and a continuous monitoring program. The primary focus of this section and associated procedures is to provide the clinical microbiologist with guidance and information for use in preparing for and responding to a suspected or confirmed bioterrorism event. Critical issues addressed herein include the types of bioterrorism events; laboratory capacity; laboratory safety, including the packaging and shipping of biological materials, especially infectious substances; and diagnostic testing protocols for those biological agents targeted as being most likely to be released in an event because they can be easily disseminated or transmitted person to person, cause high mortality with the potential for major public health impact, cause public panic and social disruption, and require special action for public health preparedness (R. Timperi, personal communication). These agents, currently classified as category A agents, include (i) Bacillus anthracis, the agent of anthrax; (ii) botulinum toxin, produced by Clostridium botulinum; (iii) Brucella spp., the agents of brucellosis; (iv) Yersinia pestis, the agent of plague; (v) Francisella tularensis, the agent of tularemia; (vi) Burkholderia mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis; (vii) staphylococcal enterotoxin B, produced by Staphylococcus aureus; and (viii) variola virus, the agent of smallpox. However, it is important to realize that any microbial agent can potentially be used in the commission of a biocrime or act of terrorism.
Chapter 16.2 : Levels of Laboratory Safety
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The importance of laboratory safety must be emphasized. Laboratory workers have always been shown to be at risk for laboratory- acquired infections. Such infections include typhoid fever; Q fever; cholera; glanders; brucellosis; tetanus; tuberculosis; tularemia; shigellosis; salmonellosis; infections caused by streptococci, Chlamydia spp., and Neisseria meningitidis; and viral infections, such as those caused by the hepatitis viruses, arboviruses, and many others ( 1 , 2 , 4 , 6 – 13 ). Interestingly, these infections have occurred in association with documented laboratory accidents in only 16% of the cases ( 13 ), of which 9.5% resulted in death ( 12 , 13 ). No national monitoring of laboratory-associated infections exists, but one estimate derived from recent surveys suggests a rate of one to five infections per 1,000 employees ( 5 , 10 , 14 ). Harding and Byers have reviewed laboratory-associated infections recently ( 3 ).
Chapter 16.3 : Packing and Shipping Select Agents
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The U.S. Department of Health and Human Services regulates and the CDC oversees the possession, use, and transfer of certain specifically listed biological agents and toxins (called “select agents”) that have the potential to pose a severe threat to public health and safety. The Select Agent Program (http://www.cdc.gov/od/ sap/) oversees all activities with select agents and registers all U.S. laboratories, persons, and other entities that possess, use, and/or transfer select agents.
Chapter 16.4 : Anthrax—Bacillus anthracis
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Bacillus anthraci., the etiologic agent of anthrax, is classified as a category A agent because of its suitability for and likelihood of use in an attack or biocrime. Disease occurs most frequently in herbivorous animals (e.g., cattle, sheep, and goats), which acquire the endospores from contaminated soil. Human disease is less common and results from contact with infected animals or with commercial products derived from them, such as wool and hides. Infection can occur in one of three forms, depending on the route of acquisition. (i) Cutaneous anthrax, responsible for >95% of naturally occurring cases, is initiated when spores of B. anthraci. are introduced into the skin through cuts or abrasions, such as when handling contaminated wool, hides, leather, or hair products (especially goat hair) from infected animals ( 11 , 19 ). There are a few case reports of transmission by insect bites, presumably after the insect fed on an infected carcass ( 1 , 16 , 17 ). This form is rarely fatal following appropriate antimicrobial therapy. (ii) Gastrointestinal anthrax may occur 1 to 7 days following the consumption of contaminated under-cooked meat from infected animals. Pharyngeal lesions may also occur from ingestion of contaminated food. Mortality in both forms is high ( 16 ). (iii) Inhalation anthrax results from the inhalation of B. anthraci. spores. Though treatable in its early prodromal stage, mortality remains extremely high if antimicrobial treatment is not initiated within 48 h of the onset of symptoms ( 20 ). A single case of inhalation anthrax should alert all health care workers to the possibility of a bioterrorism event ( 4 ). Person-to-person transmission of inhalational anthrax has not been confirmed ( 2 , 7 ).
Chapter 16.5 : BotulinumToxin—Clostridium botulinum
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Clostridium botulinu. is an anaerobic gram-positive rod that produces spores and is ubiquitous in soil and marine sediments throughout the world. Botulism is a neuroparalytic illness resulting from the action of a toxin produced by strains of C. botulinum. These toxins are extremely hazardous to humans, requiring only a minute quantity to cause profound intoxication and death. There are four distinct forms of botulism: (i) food borne, (ii) wound, (iii) infant, and (iv) adult or child. Food-borne botulism, although the most common form, is relatively rare but often fatal. This form of botulism results from the ingestion of food items containing the preformed toxin. The clinical diagnosis of food-borne botulism can be confirmed by isolating the organism from the feces of the patient. Isolation of the organism from remnants of the consumed food item does not provide confirmatory evidence of botulism in the absence of other supporting laboratory data. The demonstration of botulinum toxin in patient feces or serum or in the suspected food item supports the clinical diagnosis. Wound botulism occurs following the colonization of the wound by C. botulinum. Multiplication of the organism may result in the production of toxin. Confirmation requires demonstration of the organism and toxin in serum, feces, or material from the wound. Infant botulism results when ingested spores germinate within the intestinal tract, causing toxin production. Confirmation of infection is dependent on the demonstration of botulinum toxin in the feces.
Chapter 16.6 : Brucellosis—Brucella spp.
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Brucella is a fastidious, intracellular, aerobic, small gram-negative coccobacillus. Brucellosis is a zoonotic disease caused by four species that are recognized as human pathogens: Brucella abortus (cattle), B. melitensis (goats, sheep, and camels), B. suis (pigs), and B. canis (dogs). There have been rare reports of naturally acquired infections of humans with a marine mammal-associated species of Brucella ( 11 ). These isolates are most closely related to B. suis.
Chapter 16.7 : Plague—Yersinia pestis
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Yersinia pestis is the causative agent of plague, an acute febrile infectious disease with a high fatality rate ( 16 ). Plague may present in three forms: bubonic, pneumonic, and septicemic ( 13 ). Bubonic plague is characterized by sepsis that is accompanied by the sudden onset of fever, chills, weakness, headache, and the formation of buboes and swelling of regional lymph nodes of the groin, axilla, or neck. Septicemic plague is basically the same as bubonic plague but without the swelling of the lymph nodes. Pneumonic plague, the most deadly form of the disease and the form that can spread rapidly among susceptible individuals, presents as fever and lymphadenopathy with cough, chest pain, and often hemoptysis. Secondary pneumonia from hematogenous spread of the organisms can occur, or the organism can occasionally be passed by aerosols from human to human as primary pneumonic plague.
Chapter 16.8 : Tularemia—Francisella tularensis
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Francisella tularensis is a tiny (0.2- to 0.5- by 0.7- to 1.0-μm), pleomorphic, nonmotile, fastidious, gram-negative, facultative intracellular coccobacillus. F. tularensis can be divided into two subspecies, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B), based on virulence testing, 16S sequencing, biochemical reactions, and epidemiological features. Type A and type B strains are highly infectious (e.g., require only 50 to 100 organisms to cause disease) and are the principal agents of tularemia, a zoonotic plague-like disease distributed only in the northern hemisphere ( 3 , 4 ). In the United States, the principal reservoir is the cottontail rabbit (lagomorph), but the disease may also be carried and transmitted by a variety of terrestrial and aquatic mammals, such as beavers, ground squirrels, muskrats, and other rodents ( 3 , 4 ). Transmission to humans can occur through the handling of infected animals; through the bites of ticks, mosquitoes, or deerfly vectors; or by ingestion of contaminated stream water. F. tularensis subsp. novicida is infrequently identified as a cause of human disease.
Chapter 16.9 : Melioidosis (Burkholderia pseudomallei) and Glanders (Burkholderia mallei)
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Burkholderia malle and Burkholderia pseudomalle are considered category B select agents since they are not as easily spread as category A organisms, nor do infections due to these organisms have mortality rates as high as those of category A organisms.
Chapter 16.10 : Smallpox—Variola Major
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Variola virus, the etiologic agent of smallpox, is considered among the highly virulent microorganisms likely to be used in bioterrorism activities. If a community exposure to variola virus occurs, the challenge for the clinical microbiology laboratory will be threefold: (i) management of clinical specimens and/or viral isolates safely and appropriately, (ii) recognition of the agent within the limitations of routine testing, and (iii) rapid notification of a potential outbreak to the proper authorities.
Chapter 16.11 : Unknown Virus
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If a community exposure to a virulent unknown virus occurs, the challenge for the clinical microbiology laboratory is threefold: (i) management of clinical specimens and/or viral isolates safely and appropriately, (ii) recognition of the agent within the limitations of routine testing, and (iii) rapid notification of the proper authorities about a potential outbreak.
Chapter 16.12 : Q Fever—Coxiella burnetii
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Coxiella burnetii is the etiologic agent of Q fever. It is a pleomorphic coccobacillus that is gram negative, obligately intracellular, and 0.3 to 0.7 µm long. There is a spore-like form, the small cell variant, which is remarkably stable in extracellular environments. A large cell variant also exists that is the vegetative, metabolically active form. Mixtures of both forms are found in phagolysosomes. There is phase variation, similar to that in Salmonella, in which the lipopolysaccharide (LPS) varies chemically as either the virulent, phase I “smooth”-type LPS or the phase II “rough” LPS, associated with avirulent C. burnetii. C. burnetii is phylogenetically related to Pseudomonas, Francisella, and Legionella, within the Legionella group of the c-Proteobacteria subdivision. It is more distantly related to Rickettsia ( 7 ).
Chapter 16.13 : Clinical Laboratory Bioterrorism Readiness Plan
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The purpose of this template is to provide a model for laboratories to use for developing a bioterrorism (BT) preparedness plan. The components of this template can be used to develop a readiness plan to meet the needs of the institution. It is not meant to be all-inclusive. Rather, it is to serve as an aid in the process of developing a specific plan for each institution.
Chapter 16.14 : Avian Influenza
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Influenza A virus is a member of the family Orthomyxoviridae. Influenza viruses are enveloped, with a segmented, singlestranded RNA genome. This family also contains influenza B and C viruses. Point mutations in the envelope protein hemagglutinin (H), referred to as antigenic drift, result in the emergence of new strains of influenza A and B viruses and the resultant annual outbreaks and epidemics. New influenza A virus subtypes emerge as the result of reassortment of H and neuraminidase (N) sequences from two different subtypes, referred to as antigenic shift. These new subtypes are responsible for influenza pandemics. There are currently 16 recognized H subtypes and 9 recognized N subtypes. While virtually all combinations of influenza A subtypes naturally infect waterfowl and shorebirds, certain subtypes infect poultry and mammalian species. Subtypes H1N1, H3N2, H2N2, and H1N2 have circulated, or are currently circulating widely, among humans. Subtype H5N1, causing highly pathogenic avian influenza, was identified in 1996 in southern China. Influenza A H5N1 is significant, though not unique, in its ability to cross normal species barriers and directly infect humans; avian subtypes H9N2 and H7N7 are also known to cause infection in humans. For this reason, testing for H5N1 virus alone is not recommended, and any unusual influenza viruses that cannot be subtyped should be referred to a public health laboratory or the CDC. Among pathogenic avian influenza virus strains, the wide geographical distribution of H5N1 in avian species and the number and severity of human infections are unprecedented. If, or when, the virus evolves into a strain transmitted readily among humans, and unless there is a dramatic decrease in the pathogenicity of the resulting virus, the result will likely be an influenza pandemic with mortality rates not seen since the 1918 pandemic.
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