John C. Christenson and E. Kent Korgenski
Although not all infectious diseases require a diagnostic laboratory intervention (eg, un-complicated 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.
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
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).
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.
Transport media are necessary to maximize pathogen survival. 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. 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.
SPECIMEN-SPECIFIC ISOLATION METHODS
Specimen Collection 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 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
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
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.
Incubation When using the continuously monitored blood culture systems, most bacterial pathogens are detected within 48 hours.12,23-29 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
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. 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 ≥104 colony-forming units (CFU)/mL of a single bacterial isolate from a clean-voided midstream urine specimen (≥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. Any bacterial growth from urine obtained by suprapubic aspiration is considered clinically relevant.
Specimen Collection and Processing 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. 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. 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%).46
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.
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.
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
A culture should be obtained from all individuals with a negative rapid antigen test.55
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.
Transtracheal aspirates are technically difficult to obtain in young children and are seldom performed. Bronchoscopy, bronchoalveolar lavage, is extremely useful in the diagnosis of Pneumocystis jiroveci and mycobacterial, fungal, and bacterial infections.
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
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. 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 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. 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.
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 is still considered one of the gold standards.77 Isolation of the organism can also be attempted.
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 patients with spontaneous bacterial peritonitis, inoculation of peritoneal fluid into Bactec bottles more than doubled the recovery of gram-negative bacteria.85
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 vitroresistance 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.
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. Detection of inducible macrolide-lincosamide-streptogramin B resistance in S aureusisolates (D-test) is easily performed using disk diffusion.92
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.
SPECIFIC TESTING 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 ≥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.
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. If high-level resistance to gentamicin is noted, resistance to other aminoglycosides is predictable.
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.
Susceptibility testing of anaerobic bacteria should be limited to laboratories with special qualifications.109
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 Micro-Scan WalkAway, and the BD Phoenix system. Advantages of automated systems are that they can be connected to the laboratory computer system, provide rapid test results, allow intralab-oratory 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
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
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.
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, broncho-alveolar 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 Histoplasmaantigen 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 Histo-plasma 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.
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 (≥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
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 ethylene-diaminetetraacetic 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.
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.
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. 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.
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.
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. 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).