Chapter 226. Diagnostic Approaches for Infectious Diseases

John C. Christenson; E. Kent Korgenski

Diagnostic Approaches for Infectious Diseases: Introduction

Although not all infectious diseases require a diagnostic laboratory intervention (eg, uncomplicated otitis media), clinicians often rely on laboratory tests to help identify a causative agent, select an appropriate antimicrobial agent, and/or assess response to therapy. It is imperative that clinicians have a basic understanding of diagnostic microbiology so they can obtain appropriate samples and order the most appropriate test for diagnosing the condition they are suspecting and selecting the most appropriate therapy (Table 226-1). Often, consultation with the microbiology laboratory is useful to determine which test would be most appropriate for the suspected disease.

Table 226-1. Useful Tips from the Microbiology Laboratory

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Table 226-1. Useful Tips from the Microbiology Laboratory

Specimen collection: If clinically possible, obtain all specimens for culture before initiating antimicrobial therapy.

Urine: In young infants, preferred method for collecting urine is bladder catheterization or suprapubic aspiration. Suprapubic aspiration has lowest potential for contamination.

Cerebrospinal fluid (CSF): First tube is least likely to be contaminated.

Broth tubes and CSF: In patients without CSF shunt, gram-positive cocci in broth tube (with no growth on agar plates) are most likely contaminants.

Central venous catheters: Send blood for culture from each lumen and each venipuncture site, especially before initiating or changing antimicrobial therapy.

Stool studies: In children hospitalized for more than 3 days who develop diarrhea in hospital, stool culture and stool for ova and parasites (O&P) exam are of low yield; do not order routinely.

Nasopharyngeal aspirates or washes: Best specimens for fluorescent antibody (FA) staining for respiratory viral pathogens.

Clostridium difficile toxin assays: Do not order in children younger than 6 months.

Susceptibility testing: Minimal inhibitory concentration (MIC) values vary, depending on the antibiotic and organism being tested. A lower MIC value does not necessarily represent a more active agent.

Susceptibility testing: Not all bacteria require a complete panel of susceptibility testing: for example, Streptococcus pyogenes (no reports of penicillin or cephalosporin resistance).

It is important to recognize that molecular biology has influenced the way in which infectious diseases are diagnosed.49 DNA and RNA amplification by PCR has become an important resource in the diagnosis of infections. PCR has been shown to be more sensitive than in situ hybridization. It can detect as few as 10 to 100 copies of nucleic acid, whereas hybridization detects only 10,000 copies. PCR has been useful for identifying slow-growing organisms such as Mycobacterium tuberculosis and Borrelia burgdorferi; Rickettsia, and, potential agents of bioterrorism.153 Molecular fingerprinting has become a useful resource in the investigation of nosocomial infections and has fostered the field of molecular epidemiology.154-156

Bacteria

Listen

Collection and Processing of Clinical Specimens

No degree of laboratory expertise can correct the error of inappropriately collected and transported specimens. The proper collection and handling of clinical specimens is as important as selecting the correct medication for treatment.1,2 Common problems with clinical specimen collection and handling include insufficient quantity, contamination, inappropriate transport conditions, and delay in transport to the laboratory.1,2 The most optimal specimen for the isolation of bacteria fluid scrapings, biopsy, or tissue samples increase the chance of recovery of the pathogen and reduce the isolation of contaminants that may mislead or result in misdiagnosis. Swabs, although commonly sent are less likely to yield useful results. Swabs are commonly sent, but they are not the most optimal specimen for the isolation of bacteria. Fluid, scrapings, biopsy, or tissue samples increase the chance of recovery of the pathogen and reduce the isolation of contaminants that may mislead or result in misdiagnosis.

Transport media are necessary to maximize pathogen survival. They are designed to prevent drying, provide minimal nutrients, and maintain a balanced physiochemical environment that prevents the oxidation and enzymatic destruction of the pathogen. Some transport media may contain antimicrobial agents that suppress normal or contaminating flora to enhance the isolation of a specific pathogen. Ideally, specimens for bacterial culture should not be stored for longer than 24 hours before processing. When specimens cannot be transported or processed immediately, appropriate alternatives are available. Holding conditions are specimen or pathogen specific. Urine can be refrigerated at 2°C to 8°C for up to 24 hours. Inoculated blood culture bottles can be held at room temperature for up to 24 hours. Specimens for the isolation of Neisseria gonorrhoeae should be inoculated onto specific media (warmed to room temperature), preferably transported to the laboratory expeditiously in a CO2-enriched transport pack (Gono-Pack, Jemec) and immediately incubated at 35°C in 5% CO2. Cerebrospinal fluid (CSF) should be transported rapidly at room temperature or at 35°C to 37°C and never refrigerated. Stool for cytotoxin assay for Clostridium difficile must be refrigerated or frozen at –70°C if not processed within 1 hour because toxin activity decays rapidly at ambient temperature. A dry swab is preferred for detection of Streptococcus pyogenes antigen from pharyngeal specimens. Dacron swabs can be used for viral and S pyogenes isolation. Culturette swab is a commonly used transport system. Frequently, transport kits are assembled and supplied by local laboratories to ensure the optimal recovery of microorganisms. Specimens from infected sites where anaerobic bacteria may be causative agents, such as brain or lung abscesses or peritoneal fluid, require special anaerobic transport medium. Transporting the fluid to the laboratory for an anaerobic culture in a syringe is not adequate. Blood culture bottles should be inoculated by the bedside with an appropriate amount of blood. Synovial joint and peritoneal fluids can also be inoculated into blood culture bottles to enhance recovery yield.3-6 Sterile screw-cap containers can be used to transport urine, feces, CSF, and other body fluids.

Various types of culture media are used within the clinical microbiology laboratory. Enriched media support the growth of fastidious bacteria. Examples include chocolate agar, sheep blood agar, and thioglycolate broth. Selective media permit the selective growth of certain groups of bacteria, while suppressing others. Example include colistin-nalidixic agar (CNA) for the isolation of gram-positive organisms. Differential media assist in distinguishing among similar groups of bacteria. MacConkey agar is both selective (permitting the growth of aerobic gram-negative bacteria only) and differential (distinguishing gram-negative bacilli by their ability to ferment lactose). A lactose-fermenter organism (purple-pink colonies) on MacConkey agar may represent Escherichia coli, whereas a nonlactose-fermenter organism (clear colonies) may represent Pseudomonas aeruginosa or Shigella. The use of these media increases the yield of recovery of certain fastidious organisms and hastens the time to identification and availability of susceptibility data.

Specimen-Specific Isolation Methods

Blood

Specimen Collection

Venipuncture sites are cleansed with 70% isopropyl or ethyl alcohol, followed by disinfection with 1% to 2% tincture of iodine, 10% povidone-iodine, or 2% chlorhexidine gluconate with 70% isopropyl alcohol (allowing it to dry for at least 1 minute, with no wiping).7 The rubber stopper of the blood culture bottle or tube is also disinfected using 70% isopropyl or ethyl alcohol. Iodine is not recommended because its use may lead to cracks. Blood culture specimens from venipuncture sites are preferred as they have a lower contamination rate than specimens obtained through a newly inserted intravenous catheter.8 The proportion of blood to broth is important. A blood-to-broth ratio > 1:5 is desirable.9 Inadequate volume is the most important cause of a false-negative blood culture, and it appears to be a common problem among pediatric patients.10 Bacteremia in children is usually quantitatively higher than in adults, but low-level bacteremia does occur. An appropriate volume of blood per bottle and number of blood cultures being submitted is critical.11,12 One to 5 mL may be an adequate sample from children, whereas 20 to 30 mL is required from adults.9 Appropriate volumes would be 1 to 2 mL in neonates, 2 to 3 mL in infants, 3 to 5 mL in children, and 10 to 20 mL in adolescents.13

Most broth culture systems contain an anticoagulant, sodium polyanetholesulfonate (SPS), 0.025% to 0.05%, which inhibits phagocytosis and serum bactericidal activity, inactivates complement, and neutralizes lysozymes and some antibiotics such as aminoglycosides. Although it may enhance the recovery of certain pathogens, SPS can inhibit the growth of others such as Neisseria, Streptobacillus moniliformis (one of the causes of rat-bite fever), and Francisella tularensis (cause of tularemia). An appropriate ratio of blood-to-broth volume dilutes SPS, thereby decreasing its natural inhibitory factors, and also dilutes any antimicrobial agent that may be present, without affecting the growth of important pathogens.

Although a single sampling may be sufficient for most patients with bacteremia, especially with Staphylococcus aureus, multiple samples are needed under certain circumstances (eg, in patients with suspected endocarditis in whom three or more samples are desirable to obtain a sensitivity of 96%, especially if the patient has received antimicrobial agents). In a more recent study in adults, as many as four blood cultures in a 24-hour period were needed to achieve a detection rate of >99%.14 The total volume of blood submitted for culture is also critical. Similar studies in children are lacking at this time. For children with indwelling intravascular catheters, at least two sets of cultures are desirable: one from the vascular catheter and another from a venipuncture site.15-17

Once blood is inoculated into a broth bottle, rapid incubation is essential. In one study, a delay of 3 hours resulted in a 25% reduction in recovery of Streptococcus pneumoniae.18

Media

Current blood culture systems were developed to increase yield, reduce the time to recovery of bacteria, and diminish technologist time. Newer systems also maximize the recovery of fastidious organisms. Commonly used systems are lysis centrifugation–direct plating or the Isolator system (Wampole Laboratories), the VersaTREK (Trek Diagnostic Systems), the BacT/Alert (bioMérieux, Inc), and the BACTEC system (Becton Dickinson Division Instrument Systems). Comparisons between systems are detailed in additional text on DVD. For the latter three systems, depending on the type of suspected pathogen, several broth formulas are available. Unfortunately, no single system is optimal for all microorganisms.

The lysis centrifugation–direct plating system consists of a sterile tube of two different sizes (1.5 and 10 mL) that contains saponin and SPS, which lyse red and white blood cells in the blood specimen and inactivate complement and immunoglobulins. Hemoglobin binds to SPS and prevents the inhibition of bacteria.19 The larger tubes are centrifuged at 300 g for 30 minutes in a fixed-angle rotor. With the use of an Isostat press, the lysate is removed and inoculated directly onto agar medium, which is incubated and examined daily for growth. Media can be selected to maximize the recovery of suspected pathogens. Many facilities use this system for the isolation of non-Candida fungi and to perform quantitative blood cultures.20,21 Unfortunately, contamination has been a problem with this system. Contamination can be decreased by careful handling and inoculation of agar under laminar flow.

BACTEC has demonstrated increased recovery of organisms and decreased time to detection, particularly for Mycobacterium. BACTEC 9240 is a continuous monitoring, “noninvasive” blood culture system that uses internal fluorescent CO2 sensors to detect the metabolic activity of bacteria. Various types of media have been developed for this system. BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Peds Plus/F bottles contain resins that may decrease the effect on bacterial growth of antibiotics. By using a differential time to positivity between a positive culture result from a central venous catheter site and a peripheral venipuncture site (Å 2 hours), the diagnosis of catheter-related bloodstream infection can be confirmed.15,22 The BacT/Alert and VersaTREK systems are two additional continuous monitoring blood culture systems with demonstrable usefulness in the detection of bacteremia and fungemia, both having a higher yield of recovery and a reduction in time to detection.23

Incubation

Older “conventional” broth bottles were incubated at 35°C for 7 days. Optimally, two bottles were inoculated; one was vented with a needle to yield aerobic conditions, and an unvented one was used to maintain anaerobic conditions. Bottles were inspected daily for macroscopic growth and subcultured blindly onto chocolate agar after 72 hours of incubation. Acridine orange staining was recommended after 18 to 24 hours’ incubation. If biphasic bottles were used, they were inverted at least twice daily for the first 2 days and then daily for 5 days. Most pediatric pathogens were isolated within 72 hours of incubation. These systems are labor intensive and continuous monitoring is not feasible, so most institutions have replaced them with newer automated systems.

When using the continuously monitored blood culture systems, most bacterial pathogens are detected within 48 hours.12,23-29 Rapid detection of pathogens leads to the prompt initiation of effective therapies; shortens hospital stays, and diminishes health care–related expenses.30 Unfortunately, contaminants such as coagulase-negative staphylococci (CONS) may also grow within this time frame but generally at a slower rate; true infections with CONS are usually detected within 15 hours, whereas contaminants usually take longer than 22 hours.31 In another study, a culture that was positive within 18 hours was 13 times more likely to contain a pathogen than a contaminant. The mean time to positive cultures for contaminants was 31.1 hours.32 Incubation for more than 5 days is not usually warranted, except in certain situations (eg, suspected fungi, Bartonella henselae,Corynebacterium, Actinomyces) as detailed further in DVD text.33

Special Pathogens

In the era of the automated blood culture system, there is no need for prolonged incubations for Brucella species. For the isolation of cell wall–deficient bacteria, hypertonic medium (containing 10% sucrose or mannitol) is required. BACTEC NR-8A and BacT/Alert could also be used. Nutritionally deficient organisms such as Abiotrophia and Granulicatella should be suspected when organisms are evident on Gram stain but fail to grow when subcultured. Broth can be subcultured onto a blood agar plate streaked with Staphylococcus aureus, or media supplemented with 0.001% pyridoxal hydrochloride and 0.05% to 0.1% l-cysteine. A pyridoxal-impregnated disk can also be used. Plates are usually incubated overnight at 35°C to 37°C in 5% CO2. The HACEK group of microorganisms (Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella), which are rare causes of bacteremia and endocarditis, will grow within the routine incubation period for automated blood culture systems.33,34

Special techniques are needed for the isolation of Bartonella species and should only be performed by laboratories familiar with culture and identification of the organism. Bartonella henselae, a rickettsialike coccobacilli, is responsible for most cases of cat-scratch disease in children. The Isolator system is used, with subsequent inoculation onto media enriched with fresh 5% rabbit blood (fresh Columbia blood agar, fresh chocolate agar, and buffered charcoal yeast extract agar), and incubated in 5–7% CO2 at 35°C to 37°C for 3 weeks.

To ensure the prompt initiation of pathogen-specific antimicrobial therapy, the rapid detection and identification of pathogens, along with susceptibility data, are critical. Direct inoculation of automated systems such as VITEK from blood culture subcultures was 22 hours faster than controls in providing susceptibility data, and 13 hours faster in providing identification of the pathogen.35

The direct detection of Staphylococcus aureus and methicillin resistance from positive blood cultures can be a valuable diagnostic and therapeutic advance. The StaphSR assay (BD GeneOhm, San Diego, CA), a real-time polymerase chain reaction (PCR) assay, was found to be 100% sensitive and 98.4% specific when detecting methicillin-resistant S aureus.36 More recently, the US Food and Drug Administration approved the use of GeneXpert, a self-contained real-time PCR system for the detection of enterovirus,37 for group B Streptococcus colonization,38 and the use of methicillin-resistant S aureus directly from blood cultures.

Germ tube tests directly from blood culture bottles to identify Candida albicans may facilitate the transition of therapy to potentially less toxic agents.39

Urine

Although clean-voided midstream urine is an acceptable specimen for culture in older children and adults, this technique is difficult in young children. The collection of urine by a bag fixed to the perineum is a poor substitute (because of contamination), especially when antibiotic therapy will be initiated after collection. In young infants, urine obtained by bladder catheterization or by suprapubic aspiration is preferred. A quantitative culture is required to differentiate significant isolates from contaminants because the distal part of the urethra is normally colonized with bacteria. A known volume of urine (0.01–0.001 mL) is inoculated by means of a calibrated loop or pipette onto agar media to permit quantification of isolated colonies. Detection of Å 104 colony-forming units (CFU)/mL of a single bacterial isolate from a clean-voided midstream urine specimen (Å 102/mL from a specimen obtained by catheter) correlates well with a probable urinary tract infection. A prolonged time in ambient temperature from collection to inoculation (>2 hours) is associated with an increase in colony counts leading to false-positive culture results. Interpretation of quantitation parameters in urine specimens from uncircumcised boys when obtained by bladder catheterization or clean void should be considered identical because both collection methods are prone to contamination.40 Any bacterial growth from urine obtained by suprapubic aspiration is considered clinically relevant. Isolation of multiple organisms with low colony counts usually indicates contamination, except when a person is unable to concentrate or retain urine or when an obstructive uropathy is present.

Gram stain or acridine orange stain of unspun urine in which at least two bacteria per high-power field of the same type are demonstrated indicates significant bacteriuria (ie, Å 105 CFU/mL).

Cerebrospinal Fluid

Specimen Collection and Processing

Because CSF glucose, protein, and cell counts or the isolation of bacteria from blood culture cannot reliably exclude the presence of meningitis, CSF must be transported to the laboratory without delay because fluid is hypotonic and cells can lyse, thereby affecting the cell count and contributing to a falsely abnormal biochemical analysis (low glucose). At room temperature, cell counts (especially neutrophils) decrease approximately 32% by 1 hour and 50% by 2 hours after collection.42 Refrigeration can render fastidious bacteria such as Neisseria nonviable. If a delay is expected, samples should be stored at room temperature or incubated at 37°C. The first tube collected should be the one sent for culture because contamination is less likely. The minimal volume acceptable for culture of fungi and Mycobacterium is 2 mL; 10 to 15 mL is preferred.

Routinely, CSF should be inoculated onto sheep blood agar and enriched chocolate agar and incubated for 4 days at 35°C to 37˚C in 5% CO2. Some laboratories have limited the use of enriched broth (thioglycolate) to patients with CSF shunts. The isolation of bacteria such as coagulase-negative staphylococci from the broth tube only (with no growth on agar plates) in patients with no prior antimicrobial therapy almost always represents a contaminant. Frequently, Propionibacterium acnes grows only from the broth tube in patients with shunt infections caused by the organism. To avoid the prolonged use of antibiotics or unnecessary changes in antimicrobial therapy based on broth growth only, routine use of broth tubes for all CSF specimens is not recommended.43,44

Centrifugation by cytospinning (2000 rpm; 350 g) maximizes pellet formation of bacteria, producing a yield on direct examination of CSF by Gram stain superior to that of unconcentrated samples.45Routinely, CSF should be inoculated onto sheep blood agar and enriched chocolate agar and incubated for 4 days at 35°C to 37°C in 5% CO2. Centrifugation, with Gram stain demonstrates organisms in 75% to 90% of untreated patients with meningitis; the yield decreases to 40% to 60% in patients who have received an oral antibiotic. An acridine orange stain, or 3,6-bis(dimethylamino)acridine, is a more sensitive technique than Gram stain for detecting bacteria, especially in patients who have received antimicrobial therapy.47,48 Patients with Streptococcus pneumoniae infection are more likely to have a positive Gram stain (90%) than are those with Neisseria meningitidis (75%).46If the Gram stain is positive but the culture demonstrates no growth at 72 hours, the culture should be held for an additional 4 days. An acridine orange stain, or 3,6-bis(dimethylamino)acridine, is a more sensitive technique than Gram stain for detecting bacteria, especially in patients who have received antimicrobial therapy.47,48

Real-time polymerase chain reaction of 16S ribosomal DNA appears to be an ideal tool for the diagnosis of bacterial meningitis, especially in those children pretreated with antibiotics. Multiple studies have demonstrated its clinical usefulness, with sensitivities similar to routine culture technology but a shorter turnaround time.49,50 The technology is highly sensitive and rapid in the detection of pathogens associated with CSF shunt infections such as Propionibacterium acnes and Staphylococcus.51

Bacterial antigen detection (BAD) is usually not useful in the diagnosis and management of meningitis. Its value appears to be limited to patients with negative Gram stains, CSF, or blood cultures; pretreated patients (antibiotics for more than 24 hours); or patients in whom the lumbar puncture is postponed because of the severity of illness.46,52

Although not yet routinely recommended, in one study the inoculation of BACTEC blood culture systems with CSF led to the rapid isolation of pathogens such as Streptococcus pneumoniae.53

Respiratory Tract

Specimen Collection

Throat swabs for the detection of Streptococcus pyogenes (group A streptococcus) is the most common respiratory tract specimen sent for culture. Proper collection of the sample by swabbing the tonsillar surface, posterior pharyngeal wall, and opposite tonsillar surface, while avoiding the tongue and saliva, affects the yield of the culture. The isolation and reporting of “all” oropharyngeal organisms from a throat swab is of limited clinical value and may lead to the treatment of nonpathogenic bacterial in persons with nonbacterial causes of pharyngitis. An exception would be the young child with cystic fibrosis in whom the isolation of Pseudomonas aeruginosa from a deep pharyngeal culture may be clinically relevant.

Rapid Tests for Group a, Beta-Hemolytic Streptococci

Routine culture on agar plates requires 24 to 48 hours to detect group A streptococci and may result in a delay in therapy for some patients. Rapid detection assays for streptococcal antigen may yield results in 10 to 70 minutes. The specificity of various tests is 62% to 100%, but the sensitivity is lower (52–99%).54 Results of rapid antigen tests are influenced by the skill, experience, and expertise of the person obtaining the throat swab and performing the assay. Although initial studies performed in microbiology laboratories demonstrated a higher sensitivity for most antigen detection assays, performance in other settings has shown variable results. Rapid antigen tests using optical immunoassay (OIA) have consistently demonstrated sensitivities and specificities higher than other assays.

A culture should be obtained from all individuals with a negative rapid antigen test.55 Although the sensitivities of rapid antigen assay increase with severity of illness, the sensitivity of many assays is low enough to merit culture.56 Positive tests do not require confirmation or verification by culture.57 In addition, the isolation of Streptococcus pyogenes on an agar plate allows for the antimicrobial susceptibility testing for macrolide resistance.

New technology using chemiluminescent single-stranded DNA probes and one-rapid-cycle real-time polymerase chain reaction have demonstrated high specificity and good sensitivity. Unfortunately, the tests are difficult to perform, costly, and not suitable as point-of-care tests.58-60

Antigen detection assays have been used to detect the presence of group A streptococci, such as in pyogenic arthritis, cellulitis, and parapneumonic effusions, at extrapharyngeal sites. Abbott TestPack Plus Signify Strep Atest (Abbott Laboratories) and Directigen 1-2-3 (BD Biosciences) have shown high sensitivity when testing synovial fluid, soft tissue aspirate, and pleural fluid.61 Specificity rates were also high (99–100%).62

Special Specimens

Tympanocentesis and sinus aspiration for culture are extremely useful in special situations (eg, immunocompromised patients, patients with intracranial complications, and those who fail to respond to antimicrobial therapy). Data are conflicting regarding the validity of culture of the nasopharynx in predicting the pathogens of sinusitis and otitis media, and routine use is not indicated.

Collection of sputum from children with lower respiratory tract infections is technically difficult. Aspiration of deep pharyngeal/tracheal secretions (with a Luken trap) is used by many. In older children, sputum can be a valuable specimen. The presence of 10 or more squamous epithelial cells per low-power field is highly suggestive of oropharyngeal contamination, and the specimen should not be processed. Conversely, the presence of more than 25 white blood cells per low-power field denotes an adequate specimen. Gram stain should be used to aid in the interpretation of isolates from culture.

Transtracheal aspirates are technically difficult to obtain in young children and are seldom performed. Bronchoscopy, especially a quantitativebronchoalveolar lavage specimen or protected brush, is extremely useful in the diagnosis of Pneumocystis jiroveci and mycobacterial, fungal, and bacterial infections. Isolation of Å 104 CFU/mL is predictive of the pathogen of infection, as proved by biopsy.63-65 More studies in children are needed.

Special Pathogens

Isolation of organisms belonging to the Burkholderia cepacia complex from the sputum of patients with cystic fibrosis requires the use of selective media. Either B cepacia selective agar (BCSA) or oxidative-fermentative base–polymyxin B–bacitracin–lactose (OFPBL) medium can be used.66,67 BCSA appears to be superior to OFPBL in its ability to support the growth of B cepacia complex. Genovar identification and classification require PCR-based assays performed in laboratories specializing in this type of molecular typing.

Currently available diagnostic tests for Bordetella pertussis have variable sensitivity, depending on the stage of the disease, level of immunization, adequacy of collection and transport, and diagnostic method.68 When properly performed, culture is superior to direct immunofluorescence assay (DFA) on nasopharyngeal secretions. Serologic tests by enzyme immunoassay (EIA) (B pertussis–specific immunoglobulin IgA, IgG, and IgM) are sensitive but difficult to interpret and require acute and convalescent sera. PCR assays may provide higher sensitivity with a quicker turnaround than culture.69,70 A combination of culture, serology, and PCR provide a greater sensitivity when performed on individuals with cough illness.68,71,72

Stool and Other Gastrointestinal Secretions

Bacterial Enteric Pathogens

Most children with diarrhea have an uncomplicated course that resolves within days without antimicrobial therapy. These children rarely require laboratory testing.Infants and children with fever and/or bloody diarrhea may require specific antibacterial therapy or need close monitoring for complications such as hemolytic uremic syndrome. Stool specimens are submitted to laboratories for the routine isolation of Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7. When clinically and epidemiologically indicated, the isolation of other bacterial causes such as Plesiomonas, Aeromonas, Yersinia, and enteroaggregative E coli can be attempted. Stool specimens can also be examined for the presence of shigalike toxin from non-O157:H7 enterohemorrhagic E coli isolates.

Stool is inoculated onto various selective and differential agar media to facilitate the recovery and identification of enteric pathogens.

The routine identification of all aerobic organisms isolated from stool specimens is of no clinical utility and results in wasted technologist time and hospital charges. At times, clinicians may inappropriately treat Pseudomonas aeruginosa or Candida albicans because it was isolated as a predominant organism, but the isolation of either organism usually reflects prior antibiotic use.

The yield of stool cultures (and examinations for ova and parasites) in persons with onset of diarrhea more than 3 days after hospitalization is extremely low (< 1%). In these patients, nosocomial pathogens such as rotavirus, Clostridium difficile, and norovirus are more likely. Institutions should educate clinicians not to order “routine” stool cultures on these patients, or the laboratory should implement rejection criteria where the test would not be performed if ordered. These strategies result in significant financial savings to the institution and shortened hospital stays, as well as eliminating the wait for laboratory results of limited clinical value.44,73

Clostridium Difficile

In most laboratories, testing for Clostridium difficile toxin consists of a two-step algorithm for toxin detection. Stool is initially screened by an immunoassay for C difficile glutamate dehydrogenase antigen (C. DIFF CHEK-60, TechLab). Results are usually available within 1 hour. If this test is negative, no further testing is merited. Screen-positive specimens undergo toxin detection using cell culture cytotoxicity neutralization assay or a rapid toxin A/B assay.74,75 Culture, with detection of toxin production by C difficile isolates, is another technique for identification of C difficile disease. Recovery of the organism has the added advantage of allowing further characterization of the isolate for pathogenic mechanisms such as binary toxin production. The use of this algorithm results in a decrease in the empiric use of antibiotics such as metronidazole and oral vancomycin by several days while waiting for test results. Colonization of the gastrointestinal tract of young infants with C difficile is common and disease is unlikely because enterocytes in young children are not susceptible to toxin. Stool specimens from young infants, especially those younger than 6 months, need not be routinely tested for C difficile.76

Helicobacter Pylori

Multiple tests are available for the diagnosis of Helicobacter pylori. Histologic examination of tissue stained with Warthin-Starry silver and hematoxylin-eosin stains is still considered one of the gold standards.77In patients with suspected treatment failure for whom antimicrobial susceptibility testing is desirable, Isolation of the organism can also be attempted. For culture, commercially available Brucella agar plates with 5% horse blood or brain heart infusion (BHI) agar supplemented with 7% horse blood is the preferred medium. Biopsy samples can be tested for the presence of H pylori by testing for urease. The 13C-labeled urea breath test and serology have been advocated as noninvasive methods of diagnosing H pylori disease in children because neither test requires endoscopy. The breath test requires some degree of cooperation for 60 minutes. This assay is highly sensitive and specific. In addition, it appears to be less costly than other tests.78-80 An antigen assay performed on stool has now replaced the urea breath test at many institutions.81 Detection of specific antibodies may not differentiate between acute or past infection, and detection of specific IgM antibody is not sufficiently sensitive to be useful.82 Although PCR may be a promising diagnostic tool for H pylori infection, the rapid urease testing and histologic examination provide reliable reproducible results. PCR may be too expensive for routine clinical use. Gastric aspiration provides a useful specimen and avoids endoscopy.83

Synovial and Peritoneal Fluid

Inoculation of synovial fluid and peritoneal fluid into BACTEC blood culture bottles results in a higher recovery rate of bacteria than with conventional media. In one study, Kingella kingae, a cause of septic arthritis, was exclusively detected in specimens inoculated into BACTEC bottles.3 Investigators from France found K kingae to be the leading cause of osteoarticular infections in children at their institution by using a PCR for 16S rDNA directly on osteoarticular specimens.84 In patients with spontaneous bacterial peritonitis, inoculation of peritoneal fluid into Bactec bottles more than doubled the recovery of gram-negative bacteria.85 Although none of the fluids inoculated onto conventional media yielded streptococci and enterococci, 33% of those inoculated into Bactec bottles grew the organisms. Similar findings have been demonstrated in patients with peritonitis complicating continuous ambulatory peritoneal dialysis.86,87

Antimicrobial Susceptibility Testing

A microorganism is considered to be susceptible to an antimicrobial agent if in vitro growth is inhibited at a concentration one fourth to one eighth of that achievable in the patient’s blood, given a usual dose of the agent. In vitro resistance is highly predictive of clinical treatment failure. However, in vitro susceptibility does not ensure clinical efficacy. The site of infection and accessibility by the antimicrobial agent, protein binding, route of administration, immune status of the host, and presence of an abscess requiring drainage also influence outcomes. Organisms warrant testing if susceptibility cannot be reliably predicted. Susceptibility testing of Streptococus pyogenes is seldom necessary because of its predictable antibiogram, whereas testing for Pseudomonas aeruginosa is always indicated. The test results of certain antimicrobial agents are reported routinely, but other results are reported selectively to gain information about individual and community susceptibility patterns, as well as to control inappropriate use of certain agents, limit cost, and prevent the emergence of resistant bacteria.

Standard Media and Test Conditions

A variety of methods and media have been developed for susceptibility testing and standards have been set for testing and interpretation of results by the Clinical and Laboratory Standards Institute (CLSI). Methods and interpretation are pathogen specific.

Disk diffusion, or the Kirby-Bauer test, is a standardized technique for testing rapidly growing pathogens.88 An inoculum is prepared by direct suspension of colonies to yield a standardized inoculum that is swabbed onto the surface of a Mueller-Hinton agar plate. Reproducibility depends on the log-growth phase of organisms; therefore, fresh subcultures are always required. Filter paper disks impregnated with a standardized concentration of an antimicrobial agent are placed on the surface, and the size of the zone of inhibition around the disk is measured after overnight incubation. Classification of zone diameters into susceptible, intermediate, or resistant categories is accomplished by following CLSI interpretive guideline tables.

Another commonly employed susceptibility method is broth microdilution (MIC).93 This system allows a quantitative measurement of in vitro activity. The use of concentrations around the breakpoint MICs for resistance allows testing of more agents with fewer wells. Usually, five to eight concentrations representing therapeutically achievable ranges are tested against each organism, or one to three concentrations are used to determine activity at the breakpoint MIC. Because the exact MIC is not known, only qualitative assessment of susceptibility is determined. One must be careful with the interpretation of these values because they apply to a specific organism, the likely site of infection, the concentration of the organism at the site, and the type of antimicrobial agent being used.

The E-test (AB Biodisk) is a method that integrates disk diffusion to determine an actual MIC value and that provides accurate, reproducible results. It also allows a simple methodology for testing of anaerobes99 and fastidious bacteria.100-102 An impervious inert strip carries a marked, continuous concentration gradient of a predefined antibiotic consisting of more than 15 twofold dilutions. After incubation on seeded agar, the MIC is read at the edge of the zone of inhibition as it intersects the strip. There is good agreement between disk diffusion, agar dilution, and broth microdilution.103 This technique is especially useful for testing the susceptibility of Streptococcus pneumoniae to penicillin.104-107

When interpreting MIC results, lower MIC values for a specific antimicrobial agent do not imply that the agent is more active than one with a higher value when both are considered susceptible for that organism. Because these MIC values will differ between organisms and antimicrobial agents, comparisons deserve caution.

Automated Systems

Automated antimicrobial susceptibility testing can provide results in 2 to 18 hours.114 Systems commonly used in the United States are the BioMerieux VITEK2 System, the Dade MicroScan WalkAway, and the BD Phoenix system. The VITEK2 system may provide identification as early as 2 hours (susceptibility as early as 6 hours) after inoculation. Plastic reagent cards containing small wells or microcuvettes allow the simultaneous testing for many different antimicrobial agents. Growth is detected by means of a densitometer. It uses turbidimetrically determined kinetic measurements of growth to compute MIC values by regression analysis. The Dade MicroScan WalkAway system can provide results between 4.5 and 18 hours of incubation. The BD Phoenix System can accommodate up to 100 simultaneous tests. An indicator is added to the broth of each isolate, and redox indicator measures bacterial growth. Reliable results can be available after approximately 10 hours of incubation.115,116Advantages of automated systems are that they can be connected to the laboratory computer system, provide rapid test results, allow intralaboratory and interlaboratory standardization, are less labor intensive, and have the potential of artificial intelligence for data review. Systems can also be too restrictive for some laboratories. Panels and cards are formatted by the manufacturer with predetermined antimicrobial agents. These may not match the hospital pharmacy formulary. Systems are not appropriate for all organisms; unacceptable results (usually falsely susceptible test results) can occur for fastidious bacteria such as P aeruginosa, S pneumoniae, enterococci, Stenotrophomonas maltophilia, and coagulase-negative staphylococci.114 The initial capital investment may be prohibitive for some facilities, and a backup system is always needed in case of system failure.117,118

Specific Testing Methods

Testing of staphylococci for methicillin (oxacillin) resistance is best achieved by detection of the mecA gene by real-time polymerase chain reaction (PCR) or its product, penicillin-binding protein (PBP) 2a by latex agglutination (LA). Both tests demonstrate a 100% correlation.89,90 The LA test requires approximately 15 to 20 minutes to perform. Real-time PCR for mecA gene can rapidly detect methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles with a high degree of sensitivity and specificity.36,91 For smaller laboratories, a Kirby-Bauer test using a cefoxitin disk is a simple and inexpensive method for detecting methicillin resistance. Disk diffusion is still useful in testing staphylococcal isolates. Testing can be performed on TMP-SMX and other antimicrobials commonly used for treatment of MRSA infections that may not be available on automated system panels. Detection of inducible macrolide-lincosamide-streptogramin B resistance in S aureus isolates (D-test) is easily performed using disk diffusion.92

Inhibitory zones < 20 mm require confirmation using minimal inhibitory concentration (MIC) methodology. Because penicillin-intermediate and penicillin-resistant strains are common, blood and CSF isolates should be tested directly by MIC methods.

An oxacillin disk (1-μg disk on Mueller-Hinton blood agar) can be used as a screening test for penicillin resistance of Streptococcus pneumoniae. An inhibition zone of Å 20 mm is suggestive of β-lactam susceptibility. β-lactamase production is the most frequent mechanism of resistance with Haemophilus species, whereas ampicillin resistance mediated by an alteration in protein binding is relatively uncommon and requires MIC or disk diffusion testing for detection.

The extended-spectrum β-lactamases (ESBLs) produced by certain strains of Klebsiella species and Escherichia coli have been responsible for treatment failures with extended-spectrum cephalosporins. ESBL-producing isolates are resistant to all penicillins and cephalosporins, as well as aztreonam. ESBLs have also been detected in isolates of Salmonella, Pseudomonas aeruginosa, Enterobacter, and Citrobacter. Currently, no reliable test is available for detecting ESBLs in organisms other than Klebsiella species, E coli, and Proteus mirabilis. ESBL-producing isolates can be detected by testing specific ESBL screening drugs and confirmatory testing using cefotaxime and ceftazidime with and without clavulanic acid.94

Routine testing of Neisseria gonorrhoeae isolates is limited to detection of β-lactamase. Any other testing should be referred to public health or other specialty laboratories.95

Testing of β-lactam agents requires a strictly standardized inoculum (5 × 105 CFU/mL for MIC and 108 CFU/mL for disk diffusion testing); an increased inoculum falsely increases the MIC.96

Carbapenemases in Klebsiella pneumoniae (KPC) are now being reported more frequently in parts of the United States. These are difficult to detect, and automated systems may fail to consistently detect their presence. Clinical failures with imipenem, meropenem, and ertapenem may suggest the presence of KPC.97

Testing of enterococci for high-level resistance to gentamicin and streptomycin is useful to predict synergistic bacterial killing when combined with a β-lactam or vancomycin. Gentamicin- and streptomycin-containing agar or MIC wells (500 Âµg/mL gentamicin, 2000 μg/mL streptomycin) is used to detect high-level resistance.98 If high-level resistance to gentamicin is noted, resistance to other aminoglycosides is predictable. High-level resistance to streptomycin is specific only to this agent. Incubation for a full 24 hours is necessary (48 hours for streptomycin).

Testing for drug resistance among isolates of Streptococcus pneumoniae, enterococci, and Staphylococcus aureus deserves special mention. Most laboratories use a 1-μg oxacillin disk screening test to detect penicillin resistance among pneumococci as described previously. Penicillin MIC tests are then performed on any zone diameter of ⩽19 mm to determine if the isolate is indeed resistant. All CSF and blood S pneumoniae isolates should bypass the disk screening test and have MIC tests performed initially. Penicillin, cefotaxime or ceftriaxone, meropenem, and vancomycin should be routinely tested on these isolates. Interpretive criteria differ for cefotaxime, ceftriaxone, and penicillin for patients with meningitis and nonmeningitis. The laboratory rarely knows the clinical condition of the patient, so they will report both interpretations for each drug for non-CSF isolates.

Many susceptibility methods have problems detecting low-level vancomycin resistance among enterococci. Disk diffusion testing of vancomycin with incubation for at least 24 hours, using an agar dilution plate with brain heart infusion (BHI) agar with 6 μg of vancomycin per mL (vancomycin agar screen plate).108Vancomycin agar screen plate can also be used to detect vancomycin resistance in S aureus, which is commonly missed by disk diffusion and automated systems.

Despite the standardization of susceptibility testing by the Clinical and Laboratory Standards Institute and other professional organizations for common pathogens, there are many organisms that can cause serious infections in which methods are still not standardized and interpretative guidelines are not available. The choice of antimicrobial therapy for these infections is based on clinical experience published in the medical literature rather than on in vitro susceptibility testing.

Susceptibility testing of anaerobic bacteria should be limited to laboratories with special qualifications.109 Testing is indicated when failure of the usual therapeutic regimens has occurred or when the severe infection requiring long-term therapy is anticipated (eg, for brain abscesses, osteomyelitis, endocarditis, or refractory bacteremia). Agar dilution testing is the referenced standard for determination of MICs, but E-test methodology is more widely used by clinical laboratories.99

Testing of fastidious bacteria should be limited to a few specialized laboratories. For mycobacteria, an agar dilution test has been standard for testing. A standardized concentration of test organism is inoculated onto agar (ie, Middlebrook 7H10) containing the antimicrobial agent to be tested. After incubation for up to 3 weeks (35°C, CO2), susceptibility is determined by comparing growth on the antibiotic-containing medium with that on antibiotic-free medium.

The BACTEC 460TB radiometric system provided a major advance in the susceptibility testing of slow-growing Mycobacterium species. BACTEC 12B or Middlebrook 7H12 broth containing an antimicrobial agent was inoculated with the test organism and incubated for 5 days at 37°C. The growth index was compared with that of a control bottle containing no antimicrobial agent.110An automated nonradiometric system, BACTEC MGIT 960, appears to be as reliable as older methods.111-113

Antigen Detection Assays

Bacterial antigen detection assays were developed for the rapid diagnosis of bacterial infection. Unfortunately, these tests have suffered from poor sensitivity and specificity, and therefore their use is not generally recommended.119,120 A urinary (concentrated urine) antigen detection enzyme immunoassay for Legionella pneumophila serogroup 1 has been shown to be sensitive (89%) and highly specific (100%).121 An immunochromatographic assay has been shown to have a higher sensitivity than that of the EIA test, with results available within 15 minutes.122

The urine pneumococcal antigen assay (Binax NOW, Binax, Inc; Portland, ME) has demonstrated a high sensitivity in patients with pneumococcal bacteremia (95%) and lobar pneumonia (76%). Unfortunately, false-positive test results were observed in 15% of febrile children with no identifiable pneumococcal infection.123 There are also reports of individuals with positive results believed to be secondary to nasopharyngeal colonization.124 A more recent study found the test to be a useful diagnostic tool for patients with severe acute otitis media with complications.125 Although its use in a selective group of patients appears reasonable, recommendations for general use in febrile infants and children is not warranted at this time. A positive test in CSF (with a negative culture) would not alter the antimicrobial treatment because no organism would be available for susceptibility testing.

Other Organisms

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Fungi

Collection and Processing of Clinical Specimens

Specimens submitted for fungal culture are inoculated onto primary isolation media as soon as possible to ensure a high yield of recovery.1 If a delay is anticipated, the specimen can be stored in the refrigerator at 4°C to 8°C for up to 8 hours. It is important to prevent desiccation of the tissue specimen by adding a small amount of sterile 0.85% saline. Blood and CSF are stored at room temperature or 30°C, dermatologic specimens such as nail scrapings or hair clippings at 15°C to 30°C, and respiratory specimens should be processed fresh. Lysis centrifugation–direct plating may be the preferred method for recovery of fungi from blood.126,127 Although this is true for molds, the BACTEC system can detect most Candida isolates more quickly.128 Bone marrow should be submitted in a 1.5-mL Isolator tube.

Nails, hair, skin scrapings, fluids, exudates, and biopsy samples for direct microscopic examination can be prepared with 15% potassium hydroxide (with warming of the sample to accelerate the dissolution process). Most samples can also be examined by fluorescent microscopy with 0.1% Calcofluor white, a substance that binds to the chitin and cellulose of cell walls and causes bright fluorescence. Giemsa and Wright stains of blood or bone marrow specimens can reveal intracellular yeast forms such as Histoplasma capsulatum. Cryptococcal antigen testing of cerebrospinal fluid (CSF) (and blood) has replaced India ink staining.

Isolation and Identification

No single medium is appropriate for all specimens. Sabouraud dextrose (SAB) is the fungal medium most commonly used. The glucose concentration is 2.5%; higher concentrations can inhibit some fungi such as Blastomyces dermatitidis. To minimize the growth of saprophytes, media contains antimicrobial agents such as gentamicin, chloramphenicol, and cycloheximide.

For fluid specimens, large volumes (> 2.0 mL) should be obtained and concentrated through centrifugation at 1500 to 2000 g for at least 10 minutes. Tissues are minced and homogenized. Mycosel medium (BBL Microbiology Systems), which consists of SAB and cycloheximide, is recommended for isolation of dimorphic fungi. BHI agar containing gentamicin and chloramphenicol in addition to 10% sheep blood is recommended for isolation of Cryptococcus neoformans in potentially contaminated fluids such as sputum. Blood obtained in Isolator tubes should be inoculated onto chocolate agar, BHI, and inhibitory mold agar (IMA).

After isolation, moulds are frequently identified by the morphologic characteristics of spores and structures. Lactophenol cotton blue mounts are commonly used. To detect dimorphism, the organism is incubated at 25°C and 37°C to detect the presence of mold or yeast forms, spherules, or endospores, respectively.

The germ tube test, in which yeast is incubated for 2 to 3 hours at 35°C in fresh fetal calf serum, identifies Candida albicans by outgrowth of a tube (not all C albicans are germ tube positive, however). Commercial systems can be used to identify many yeasts by assimilation of carbohydrates for growth. A commonly used system is API Analytab 20C Yeast Identification System. Other frequently used systems are BBL Minitek, BioMerieux VITEK2, MicroScan, and BD Phoenix.

An antigen detection latex agglutination test for C neoformans is commercially available and has been a highly useful clinical tool. However, laboratory personnel should be aware that the disinfectants and soaps used to clean ring slides can cause false-positive test results.129 Immunoassays performed on urine (CSF, bronchoalveolar lavage fluid, and blood) can detect Histoplasmosis capsulatum and Blastomyces dermatitidis antigens. These tests are particularly useful in the diagnosis of infections in the young infant and the immunocompromised host.130-134 Caution in interpreting results is merited because cross-reactivity between fungi does occur. However, on occasion this cross-reactivity may be of diagnostic utility. Coccidioidomycosis may be diagnostic in endemic regions by using the Histoplasma antigen assay.135 Circulating Aspergillus galactomannan antigen can be detected in blood specimens from patients with suspected invasive aspergillosis by using an EIA method. Frequent screening with this assay may lead to an earlier diagnosis in many immunocompromised individuals.136 Interpretation of positive test results merit careful consideration in patients receiving antibiotic therapy containing β-lactamase inhibitors such as piperacillin-tazobactam because false-positive results have been reported.137 In addition, clinicians need to be cautious in interpreting negative results because antifungal therapy appears to decrease sensitivity of the assay.138 Invasive aspergillosis involving the lungs can be diagnosed by performing galactomannan assay from bronchoalveolar lavage (BAL) fluid.139,140 Pulmonary histoplasmosis can also be diagnosed by performing a Histoplasma antigen assay on BAL fluid. Normal saline solution should be used instead of Plasmanate to avoid false-positive results.141

Antifungal Susceptibility Testing

Because of the availability of new azole agents and echinocandins with variable activity against fungi and the emergence of resistance in clinically relevant fungi and yeasts to amphotericin B and other agents, antifungal susceptibility testing is appropriate on isolates from normally sterile sites. The addition of newer antifungal agents such as caspofungin, micafungin, anidulafungin, and voriconazole to our treatment selection has given options to clinicians to more effectively treat infections such as aspergillosis and candidiasis. Combinations of these agents may be desirable in some patients because synergy may be present. Little data exist, however, to enable correlation of test results with clinical outcomes.Unique problems of fungi are a slow growth rate, as well as dimorphism in some. The endpoint MIC for amphotericin B is easily defined, but there is overlap of MIC values for susceptible and resistant isolates. Endpoints for azoles are difficult to determine because of their unique partial inhibition of fungi. The technical aspects of testing must be carefully controlled following CLSI recommendations and guidelines to avoid inaccuracies.

Because some fungi demonstrate resistance, antifungal susceptibility testing may be more important now than ever.142 Due to potential inter- and intralaboratory reproducibility problems, this type of testing should be limited to a few specialized laboratories.143

Parasites

Examination of Feces

Collection and Preservation

Proper collection and handling of stool specimens is critical for the detection of parasites. Stool should be collected in a clean, wide-mouthed container with a tightly fitting lid. The specimen should not be contaminated with urine or water from the toilet. If transportation to the laboratory is not prompt, the specimen should be stored in the refrigerator. Dry samples are not acceptable. A minimum of three samples over a period of 7 to 10 days is generally needed for accurate testing. In a more recent study, only 72% of parasites were detected with a single specimen. Another 28% were identified with two more specimens.144 In areas of high parasite prevalence (Å 20%), a comprehensive evaluation of a single specimen may have enough sensitivity to be clinically appropriate.

Liquid stool should be examined or preserved within 30 minutes of collection, soft or semiformed stool within 1 hour, and formed stool the same day. Two commonly used preservatives are polyvinyl alcohol (PVA) and formalin. PVA is a commonly used fixative and preservative; it provides a good permanent smear and preserves the integrity of trophozoites and cysts. PVA should be handled with care because it is poisonous. Formalin is a useful preservative in general, and formalin specimens can use a concentration procedure to increase parasite detection, especially for protozoan cysts and helminth ova. Formalin is superior for helminth ova, Giardia, and Isospora belli.

Examination

Stools are examined grossly for consistency, the presence of mucus and blood, and the presence of adult worms or proglottids. Trophic amebae and flagellates are encountered more commonly in liquid and soft specimens. However, testing must be performed quickly because these organisms disintegrate rapidly at room temperature. For protozoans, trophozoites and cyst forms are observed more commonly in semiformed stools while cysts are seen in formed stools.

A direct wet mount smear can be prepared with 0.85% saline or D’Antoni or Lugol iodine. The specimen is scanned at 10× and then at 40× looking for motile organisms. Because it requires fresh stool, it is infrequently done.

Concentration of feces is important when small amounts of organisms are expected. It is designed to facilitate the recovery of protozoan cysts, coccidian oocysts, and helminth eggs and larvae.

A permanent stain smear is prepared for the identification of intestinal parasites, mainly trophozoites, using a PVA-preserved specimen. Wheatley-Gomori tissue trichrome and iron hematoxylin stains are preferred. Permanent stain allows definition of morphology and provides a slide for reference.

Detection of Cryptosporidium species can be done by special stains or antigen detection. Cryptosporidium species are not usually seen with normal permanent stains and require the use of special stains such as modified Kinyoun or Ziehl-Neelsen. Such stains can also be used to detect Isospora belli and Cyclospora.

Monoclonal antibody assays (EIAs) or direct fluorescent antibody stains for Giardia and Cryptosporidium are useful when the density of parasites is expected to be low. In specialized laboratories, fecal culture methods or coprocultures (Baerman technique) can be used to recover parasites. These techniques are useful in low-density infestation with hookworms, Strongyloides, and Trichostrongylus.

A simple way of obtaining larvae and adult worms of Enterobius vermicularis for examination is the cellulose tape method. Tape reversed on a tongue depressor is pressed against the anus in the early morning. The tape is then placed over a slide and examined under light microscopy.

Stool antigen detection assays using polyspecific polyclonal antibody are nonspecific and less sensitive in transported/stored samples with fixatives. Giardia antigen GSA 65, which is specific to Giardia lamblia and stable in the gastrointestinal tract and in fixatives, is a potentially useful antigen. Detection with a Giardia antigen detection method (Prospec T/Giardia, Alexon) appears sensitive and specific when testing symptomatic and asymptomatic individuals, with a sensitivity of 96% versus 74% for stool examination for eggs and parasites.145 Other immunoassays detect also Cryptosporidium antigens.146

Blood Parasites

Thin- and thick-film smears stained with Giemsa or Wright stain are still one of the most reliable and efficient way of diagnosing bloodstream parasitic infections caused by Plasmodium, Babesia, Trypanosoma, and Leishmania. Borrelia and Yersinia pestis can also be visualized with these stains. Unfortunately, because most of these parasites are infrequently observed in laboratories in developed countries, maintaining a sufficient level of expertise is difficult. Parasites are more easily observed in the thinner portion of thick film. Thick film should be examined for 5 to 10 minutes (about 100 fields) and thin film for 15 to 20 minutes (300 fields). A single blood sample is not sufficient to exclude a diagnosis of these parasitic diseases. Blood samples should be submitted every 6 to 12 hours. Anticoagulated blood (with ethylenediaminetetraacetic acid [EDTA]) is acceptable only if the sample is less than 1 hour old. Buffy coat preparations can be stained to visualize the amastigotes of Leishmania within monocytes. Finger-stick blood is preferred when examining blood for Plasmodium.147

Newer technology such as Binax NOW ICT malaria antigen assay compares favorably with microscopic smear examination in the diagnosis of malaria of moderate-to-high parasitemia. The assay may even differentiate between Plasmodium falciparum and non-falciparum species.148 However, sensitivity in detecting Plasmodium ovale is lower than desired.149

Other Sites and Organisms

Duodenal contents can be examined for parasites and protozoa by means of the Enterotest capsule or aspiration of fluids during endoscopy.

Trichomonas vaginalis can be observed on direct saline wet mounts from vaginal secretions or first-voided urine (or both), or from other fluids such as respiratory tract secretions from neonates.

Tissue obtained by biopsy is the preferred specimen for certain severe parasitic infections such as toxoplasmosis and P jiroveci pneumonia. Impression smears, squash preparations, and scrapings from skin and nails are commonly submitted specimens. These specimens are stained and examined microscopically. Giemsa and/or trichrome and methenamine silver stain of the lung, small intestine, skin, or brain can reveal organisms such as P jiroveci, Toxoplasma gondii, Leishmania, Naegleria, or Entamoeba. Modified acid-fast staining of the small intestine allows visualization of Cryptosporidiumparvum, I belli, and Cyclospora.

Rickettsiae

Rickettsial organisms are difficult and hazardous to recover and also difficult to visualize with light microscopy. In some patients, the morulae (inclusion bodies) of Ehrlichia and Anaplasma can be observed within the monocytes and neutrophils, respectively. Immunofluorescent stain of biopsy samples is useful. Most diagnoses are based on clinical features and confirmed with serologic tests.150 The most commonly used tests are the indirect fluorescent assay and the microimmunofluorescent test. Specific diagnostic tests are discussed in Chapter 294.

Viruses

The development of molecular diagnostic testing with PCR has allowed the clinician a better understanding of the epidemiology and clinical presentations of many viral agents, provided an assessment of antiviral therapeutic regimens, and defined the natural course of many viral diseases and outcomes after therapy. In addition, newer human pathogens such as bocavirus, metapneumovirus, Hantaviruses, and SARS coronavirus can be identified by PCR.

Although acute and convalescent serologic testing has high sensitivity and specificity, it is not clinically useful in the immunocompromised host who is incapable of mounting an appropriate immune response. In addition, because a convalescent specimen is needed in most cases, it has limited utility as a rapid diagnostic test.

In addition to PCR, other tests are still clinically useful. Among these are enzyme immunoassays, shell vial tissue cultures, fluorescent staining, and microarrays.157

Stool specimens can be tested for rotavirus and adenovirus 40/41 antigen using a commercially available EIA kits. EIAs are frequently used as rapid diagnostic assays for respiratory viruses such as respiratory syncytial virus (RSV) and influenza. It is important that clinicians be aware that the predictive values of EIA influenza testing vary significantly during the year, with higher positive predictive values (PPV) during the peak influenza season and low PPV in times of low prevalence. Many of these tests are point-of-care tests. The appropriate use of these assays may diminish the inappropriate use of antibacterial agents in persons with respiratory infections, resulting in a reduction in health care charges by shortening hospitalization stays and potentially preventing the development of bacterial resistance.158,159

A useful diagnostic technique is fluorescent antibody (FA) staining. Well-trained technologists can identify multiple respiratory viruses such as RSV; influenza A and B; parainfluenza 1, 2, and 3; adenovirus; and metapneumovirus from a single specimen. Results can be available within 1 to 2 hours.160-162 Because false-positive and false-negative results do occur, a high level of technical skill and experience within the laboratory is important. In addition, specimen collection and quality is just as important. Nasopharyngeal aspirates or washes provide sufficient epithelial cells for reliable testing. Smaller laboratories or clinics may prefer to use other technologies because FA testing requires a fluorescent microscope. RT-PCR is also excellent for testing for respiratory viruses.163,164 A more recently released commercially approved diagnostic PCR, Respiratory Viral Panel (Luminex Corporation) allows the detection of multiple respiratory viruses, including human metapneumovirus, rhinovirus, and adenovirus.

Shell vial culture technology is still being used in many diagnostic laboratories. It compares favorably with PCR, especially when combined with DFA testing. In addition, it allows the isolation of the virus, which permits serotyping for epidemiologic surveillance and outbreak investigations.157

Fluorescent antibody staining of antigens obtained from vesicles or pustules for HSV and varicella zoster virus (VZV) is diagnostic and can have a rapid turnaround time. Because VZV is difficult to grow in tissue culture, FA and PCR testing can be quite useful. FA staining of buffy coat cells from blood is a rapid method for quantifying cytomegalovirus (CMV) in high-risk persons with suspected disease.165 The higher the degree of antigenemia, the more likely it is that the patient has disease. Response to therapy can also be monitored. Unfortunately, this assay requires the presence of white blood cells, thus limiting its utility in the immunocompromised neutropenic host. This technology is not considered point of care.

Quantitative PCR assays allow clinicians the ability to monitor virologic response to antiviral therapies for HIV, CMV, hepatitis B and C, and Epstein-Barr virus. Specific discussion of the utilization of PCR for diagnosis and therapy is found in the chapters focused on each viral pathogen. Identification of persons with HSV encephalitis, a condition in which few persons would ever have a positive CSF viral culture (~10%), can now be achieved in a reliable and prompt manner. A positive PCR at the end of therapy predicts poor outcomes and may represent a need for continuing acyclovir therapy. PCR can also be used to identify other causes of meningoencephalitis in children such as enteroviruses, arboviruses, and Mycoplasma pneumoniae. Laboratories have to be careful to avoid contamination within their facilities because this may lead to false-positive PCR results.

The immunocompromised host, such as those with cancer or solid organ and stem cell transplant with suspected infections, have posed a clinical dilemma over the years. With PCR, we are now able to diagnose BK virus as a cause of urinary bleeding and kidney transplant rejection, Epstein-Barr virus–induced lymphoproliferative disease, or fever of unknown origin, and CMV as a cause of fever with hepatic dysfunction. Quantitative PCR in patients with chronic hepatitis B and C can be monitored for response to therapy.

PCR can be performed on most tissues or body fluids. Cerebrospinal fluid should be stored frozen if a delay is expected in processing the specimen for enterovirus or HSV PCR. Respiratory specimens need to be submitted to the laboratory in viral transport medium (Microtest M4 or UTM).

References

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Other Organisms

Fungi

Collection and Processing of Clinical Specimens

Isolation and Identification

Antifungal Susceptibility Testing

Parasites

Examination of Feces

Collection and Preservation

Examination

Blood Parasites

Other Sites and Organisms

Rickettsiae

Viruses

References

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