Symposium on Anti-Infective Therapy
Diagnostic Microbiology and Therapeutic Drug Monitoring in Pediatric Infectious Diseases Michael R.]acobs, M.B., B. Ch., Ph.D., M.R.C. Path., F.F. Path. (S.A.), D.P.H., D.T.M. & H.* and Carolyn Myers, Ph.D.t
Optimal management of infections is based on isolation of the causative microorganism(s) and on accurate antimicrobial susceptibility testing. Recent improvements in commercially available products have greatly facilitated these diagnostic procedures and techniques for identification and susceptibility testing of common pathogens are now available in all laboratories. COLLECTION AND PROCESSING OF SPECIMENS Accurate laboratory results require appropriate collection, transportation, and processing of the clinical specimen. The ideal specimen is (1) from a normally sterile body site; (2) not contaminated by indigenous organisms of the skin or mucous membrane; (3) promptly transported to the laboratory in a manner designed to protect fastidious organisms; (4) accompanied by relevant information about the patient (including clinical diagnoses and examinations required); and (5) promptly examined and cultured on appropriate media on receipt by the laboratory.l 9 · 41 An adequate quantity of material is essential, particularly if culture for different groups of organisms is required. Whenever possible, specimens should be collected before the institution of antimicrobial therapy, and use of topical anesthetics or preservative-containing solutions should be avoided. 19 Specimens from different body sites require specific collection and
*Assistant Professor of Clinical Microbiology and Medicine, Case Western Reserve University School of Medicine, and Director of Clinical Microbiology, University Hospitals of Cleveland, Cleveland, Ohio tSenior Research Associate in Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio
Pediatric Clinics of North America-Yo!. 30, No. 1, February 1983
R. JACOBS AND CAROLYN MYERS
processing. In the respiratory tract, various sites of infection occur. Pharyngitis can be evaluated using a throat swab for bacterial culture and throat swabs or washings for viral isolation. Adequate specimens from lower respiratory tract infections are difficult to obtain, particularly in children; sputum is often inadequate, and invasive procedures such as bronchoscopy, transtracheal aspiration, and lung biopsy may be required. Specimens for viral isolation require preservation in a viral transport medium, such as Hank's balanced salt solution with gelatin. 22 For collection of urine, clean-voided midstream specimens are essential to minimize contamination. Suprapubic aspiration is useful in infants. Such specimens should be clearly identified, as any bacterial growth is significant. Because urine is a good culture medium, specimens should be refrigerated if not planted within an hour of collection. For the diagnosis of bacteremia, blood should be collected aseptically and inoculated into suitable culture media at a ratio of up to I:IO of blood to culture medium. Collection of small volumes of blood at intervals is preferable to a single collection of a large volume inoculated into several sets of cultures. 4 Pus and purulent exudates should be submitted in an air-free syringe or in a suitable transport medium to ensure survival of anaerobes. Cerebrospinal fluid must be collected aseptically and cultures planted as soon as possible, preferably at the bedside. An adequate volume of cerebrospinal fluid is required, particularly if mycobacterial and fungal cultures are requested. In diarrheal diseases, feces should be submitted in preference to rectal swabs, and culture for specific pathogens such as Vibrio cholerae, V. parahaemolyticus or Campylobacter fetus requested if indicated on the basis of travel or food ingestion. Genital or other specimens suspected of containing gonococci require immediate plating on Thayer-Martin medium or submission to the laboratory in a suitable transport medium. DIRECT EXAMINATION OF SPECIMENS Specimens can be examined macroscopically or microscopically, and products or antigens of infecting organisms can be detected by immunologic, chromatographic, and other methods. In addition, specimens can be examined for inflammatory cells and various chemical components, including protein and glucose. Macroscopic examination of specimens can provide valuable information on the presence of blood, pus, and helminths, as well as the clarity of specimens such as cerebrospinal fluid and urine, and the odor of pus containing anaerobes or Pseudomonas aeruginosa. In addition, macroscopic selection of purulent portions of specimens such as sputum and feces is essential to enable representative material to be examined microscopically. Microscopy Microscopic examination of specimens often provides diagnostic information. Light microscopy is used to examine wet mounts of specimens for
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
organisms and inflammatory cells, although these are best seen using phase contrast or dark field techniques-for example, to visualize spirochetes and protozoa. Demonstration of Cryptococcus neoformans can be performed by mixing a drop of India ink with a drop of specimen such as cerebrospinal fluid to provide a negative stain to outline the capsule of the organism. Potassium hydroxide wet mounts are useful for detecting dermatophytes in skin scrapings and hair. Staining preparations of fixed smears is the most common microscopic procedure performed. Gram-stained smears in untreated acute bacterial meningitis due to Haemophilus injluenzae, Neisseria meningitidis or Streptococcus pneumoniae are positive in 60 to 80 per cent of cases. 19 Smears of cerebrospinal fluid are best prepared from centrifuged deposits--centrifugation at 1000 g for 15 minutes or longer is required to sediment H. influenzae and other organisms in early infections before inflammatory cells are present. 19 Gram-stained specimens of other body fluids can be helpful. Examination of specimens from the lower respiratory tract is useful in the initial management of pneumonia. Material is representative of sputum when inflammatory cells are seen; if oral squames and a wide variety of organisms are seen, the specimen represents saliva, and culture will not be of diagnostic assistance. Diagnosis of pneumonia due to pneumococci, H. influenzae, Staphylococcus aureus, and enterbacteriaceae can be suggested by the gram-stain appearance. Gram-staining of a drop of well-mixed urine is suggestive of significant bacteriuria if 2 or more bacteria are seen per oil immersion field. 42 Mycobacteria stained with various stains resist acid decolorization, and various acid-fast stains are available for this group of organisms. The most common mycobacterium seen in specimens by acid-fast staining is M. tuberculosis. Various acid-fast stains are used, such as the Ziehl-Neelsen stain using hot carbol fuchsin, the Kinyoun stain using cold carbol fuchsin, and the auramine-rhodamine stain. With carbol fuchsin, mycobacteria stain red; while with auramine-rhodamine, mycobacteria fluoresce bright yellow against a dark background under suitable fluorescent illumination. Fluorescent antibody staining is extremely useful for demonstrating the presence of various pathogens. Specific fluorescein-conjugated antisera are used to stain smears, which are then examined on a fluorescence microscope for bright green fluorescing organisms. Organisms identified in this way include Bordetella pertussis, Francisella tularensis, and Le-
gionella pneumophila. Other staining methods can be used when indicated, for example, silver stains of biopsies for fungi and Pneumocystis carinii, trichrome stain of fecal smears for protozoa, and Giemsa stain of eye exudates for chlamydia} inclusions and of blood films for parasites such as Plasmodium and Trypanosoma species. Detection of Microbial Products A variety of techniques have been devised to detect microbial antigens, endotoxins, and metabolic products in body fluids. The only technique commonly available is the detection of antigens by various immunologic methods, and these will be discussed.
R. JACOBS AND CAROLYN MYERS
Antigens, usually capsular polysaccharides, of H. influenzae, N. meningitidis, S. pneumoniae, S. agalactiae (group B streptococcus), and Cryptococcus neoformans can be detected in cerebrospinal fluid by counterimmunoelectrophoresis (CIE), latex agglutination, or staphylococcal coagglutination. Reagents for latex agglutination are commercially available for H. influenzae and C. neoformans, and for staphylococcal coagglutination for H. influenzae, S. pneumoniae, and S. agalactiae. Counterimmunoelectrophoresis is based on antigen-antibody precipitation when negatively charged antigen moves toward the anode and the antibody moves toward the cathode. 1 Antigen and homologous antibody form a precipitin line between the anode and cathode. Antisera to H. inJluenzae (type b), S. pneumoniae (omniserum), N. meningitidis (groups A, B, C, and Y) and S. agalactiae are commercially available and can be used to detect antigen in cerebrospinal fluid. False-positive counterimmunoelectrophoresis results occur; for example, N. meningitidis type B and E. coli K1 antigens cross-react, and artifacts due to precipitated protein need to be distinguished from true lines of precipitation. S. pneumoniae types 7 and 14 polysaccharides have no negative charge and may not be detected by counterimmunoelectrophoresis unless boronic acid-containing buffers are used-these buffers, however, decrease the sensitivity for detection of other antigens. 1 Counterimmunoelectrophoresis for meningococci presents problems, as polyvalent antisera do not produce good results, and group B antisera commercially available are of very low sensitivity. High antigen concentrations can result in false-negative counterimmunoelectrophoresis results owing to prozone phenomenon and require dilution of specimens for retesting. Low antigen concentrations-for example, in urine--may require concentration of specimens. Current clinical uses of counterimmunoelectrophoresis are given in Table 1. Counterimmunoelectrophoresis of cerebrospinal fluid is particularly useful in the diagnosis of meningitis and is positive in over 90 per cent of bacteriologically proven cases. False-positives with cerebrospinal fluid are rare, and antigens can be detected in patients already treated with antibiotics. Group B meningococci, and types 7 and 14 pneumococci, however, present problems of detection. 1 Counterimmunoelectrophoresis can be useful in detecting 50 to 75 per cent of patients with bacteremic pneumococcal pneumonia using serum and concentrated urine, but only detects about 45 per cent of nonbacteremic cases. Antigen can persist in urine for as long as 4 to 6 weeks Table 1. Applications of Counterimmunoelectrophoresis Detection of Microbial Antigens Streptococcus pneumoniae Neisseria nwningitidis groups, A, C, and Y Haemophilus injluenzae type b Group B streptococcus
Antigens can be detected in Cerebrospinal fluid, serum, urine, other body fluids Cerebrospinal fluid, serum, subdural and joint fluids Cerebrospinal fluid, serum, urine Cerebrospinal fluid
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
after onset of illness, and can be useful in retrospective diagnosis. Sputum has been reported positive by counterimmunoelectrophoresis in most bacteremic patients. Prognostic implications of counterimmunoelectrophoresis include greater incidence of disseminated intravascular coagulation in pneumococcal pneumonia with positive serum counterimmunoelectrophoresis, greater incidence of subdural effusion in H. influenzae meningitis with 1 J..Lg/ml of antigen in cerebrospinal fluid (2::20 X detectable limit), and poor prognosis in meningococcal meningitis when serum counterimmunoelectrophoresis is positive. 1 Use and interpretation of counterimmunoelectrophoresis must take the above factors into consideration. The main application of counterimmunoelectrophoresis is in the diagnosis of meningitis when the gram-stain of cerebrospinal fluid is negative or when antimicrobial therapy has been started. Positive counterimmunoelectrophoresis in sputum, joint and pleural fluid, serum, and urine can be useful in confirming a diagnosis, although false-positive results do occur and urine can remain positive for prolonged periods of time. Latex and coagglutination appear to be more sensitive, less expensive, and provide results more rapidly than counterimmunoelectrophoresis. Accordingly, these techniques should become more widely used.I 1 Latex or coagglutination is performed by placing one drop of latex or staphylococcal suspension and two drops of specimen (cerebrospinal fluid, serum, etc.) in a marked area on a glass slide, and rotating the slide for 10 minutes. A positive result is obtained when the latex particles or staphylococci agglutinate and visible clumps are seen. Uncoated particles run in parallel should show no agglutination for a positive result to be valid. Enzyme-linked immunosorbent assays (ELISA) for the detection of bacterial antigens using enzyme-labeled antibodies show promise for detecting a variety of microbial antigens, but the only commercially available reagent at present is for the detection of rotaviruses. Future developments may well result in other ELISA systems becoming available for many organisms. SPECIMEN PROCESSING
Isolation of bacteria is generally carried out on agar media incubated in room air, in a 5 to 10 per cent C0 2 atmosphere or under a strictly anaerobic environment to allow the growth of different groups of bacteria. A wide variety of bacteriologic media are available, and selective and enrichment media are often required to allow the isolation of pathogens from sites containing normal flora. Examples of commonly used bacteriologic media include blood agar for growth of many common bacteria, chocolate agar for growth of more fastidious organisms such as H aerrwphilus spp and pathogenic Neisseria spp, MacConkey agar for growth of enteric gramnegative bacilli, and Thayer-Martin medium for selection of pathogenic Neisseria spp from normal flora sites. Mycobacteria can be isolated on Lowenstein-Jensen or Middlebrook
jACOBS AND CAROLYN MYERS
media, and most pathogens grow after incubation for two to three weeks. More rapid detection of mycobacteria can be achieved using a liquid medium containing 14C-labeled substrates, which are monitored radiometrically for 14C0 2 production and allow isolation within a week. Fungi can be isolated using a variety of media. Yeasts usually grow readily on blood agar after two to three days incubation, and media of low pH such as Sabourand agar can be used to decrease bacterial growth. Filamentous fungi grow slowly and require isolation on media such as Sabourand agar or yeast extract agar incubated at 25°C and 37°C. Selective agents to inhibit bacteria such as cyclohexamide and various antibiotics can be added to these media. Viruses can be isolated using animal inoculation, embryonated hens' eggs, or tissue cultures (mammalian cell cultures). Tissue cultures are generally used because they are more readily available, but not all viruses will grow in tissue cultures on primary isolation. Commonly used cell lines for tissue cultures include monkey kidney, human embryonic lung fibroblasts, and HeLa cells. Chlamydia, although bacteria, have a unique intracellular developmental cycle and form characteristic inclusions in suitable cell cultures such as treated McCoy cells. 36 Chlamydia can also be isolated in embryonated hens' eggs. Isolation procedures to detect common pathogens of various organ systems are given below. Respiratory Tract Infections
Throat Cultures. In acute pharyngitis, culture for group A streptococci is routinely performed by inoculating a blood agar plate and incubating at 35 to 37°C aerobically, or preferably anaerobically under 5 to 10 per cent C0 2 • 10 Group A streptococci grow as small colonies surrounded by a large, well-defined zone of complete hemolysis; such colonies must be differentiated from non-group A streptococci, preferably by antigenic identification (for example, by direct fluorescent antibody staining or by latex agglutination) or by presumptive identification using 0.04 U bacitracin disks. Hemolytic streptococci should be subcultured on a plate with a bacitracin disk-if a zone of inhibition around the disk is seen after overnight incubation, the test is positive for a presumptive group A streptococcus. False-positive results with bacitracin disks can occur, and antigenic identification of group A streptococci is preferred. Use of bacitracin disks on primary plates identifies only up to 65 per cent of group A streptococci, and the performance of such cultures in physicians' offices, should be discouraged unless adequate access to a microbiology laboratory is not available. Nasopharyngeal Cultures. Cultures of the nasopharynx can be used to detect carriers of group A streptococci, Neisseria meningitidis, C. diphteriae, and Bordetella pertussis, and the laboratory must be specifically informed as to which organism is being sought to enable culture on adequate media. 41 Sputum Culture. Interpretation of sputum cultures is difficult in view of the difficulty in obtaining specimens in children and contamination with saliva. Initial examination of .specimens can be done by gram-staining. Bacterial culture of sputum should be performed on blood agar incubated
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
under C02 for pneumococci and staphylococci, chocolate agar under C0 2 for Haemophilus spp. and MacConkey's agar for enteric gram-negative bacilli. Mycoplasma pneumoniae can be isolated on special media, but isolation is rarely attempted, since growth is slow and adequate material for culture often is not available. Culture for mycobacteria and fungi can be performed using suitable media. Prolonged incubation is often necessary for mycobacteria and for most fungi. Transtracheal aspirates, bronchial washings, pleural aspirates, transbronchial biopsies and open-lung biopsies are the most reliable specimens for the microbiologic diagnosis of pulmonary infections caused by bacteria, viruses, and fungi. Anaerobic cultures can be performed on transtracheal and pleural aspirates and on biopsies, and culture on specific media for Legionella pneumophila can be performed on these specimens. Urinary Tract Infections Clean-voided midstream first-morning urine specimens are ideal but are obviously difficult to obtain in infants and small children. Suprapubic aspiration is recommended. Quantitation of voided urine is required to differentiate between infection and urethral or perineal contamination of specimens. Significant bacteriuria (;:::100,000 per ml) can be detected by placing 1 ,...I of well mixed urine onto blood and MacConkey agar plates, which are incubated aerobically (calibrated 1-,.d loops are used for this purpose). Any number of organisms in suprapubically aspirated urine is significant. 42 Miniaturized culture devices are available for screening urine for significant bacteriuria by dipping these devices, which usually are coated with two culture media, into urine specimens. After incubation, quantitation can be obtained by comparing growth density with charts provided. Caution should be used with these systems, as their small culture surfaces may obscure the presence of mixed bacterial populations and they may absorb antimicrobial agents present in urine, producing a false-negative result. These devices should be considered only when laboratory access is not available or when facilities for microscopic examination are impractical or not available. Septicemia Septicemia is diagnosed by the detection of bacteremia by blood culture. Organisms are often present in the bloodstream intermittently and in low numbers, and success in the detection of bacteremia depends on the volume of blood collected and on collecting multiple specimens before the institution of antimicrobial therapy. These factors are difficult to apply in neonates and infants, because only small volumes of blood can be obtained and rapid institution of antimicrobial therapy is often mandatory. However, bacteremias in children are often of high degree and generally present little difficulty in detection. Specimens should be inoculated into two or three different liquid media, one of which provides anaerobic conditions. A volume of blood up to 10 per cent of the medium volume should be inoculated into each bottle. Cultures are incubated and inspected daily for up to seven days for evi-
R. }ACOBS AND CAROLYN MYERS
dence of growth macroscopically or by release of 14 C0 2 using the Bactec system. Blind subcultures of all aerobic bottles should be performed at some time during the incubation period, preferably during the first 48 hours. Most commercial blood-culture media contain the anticoagulant sodium polyanetholsulfonate (SPS), which is also anticomplementary and antiphagocytic, but it also inhibits the growth of meningococci and gonococci; media without SPS should be used when these organisms are suspected. Yeasts will grow in conventional blood culture media but are best isolated in biphasic solid/liquid phase media that can be incubated for up to three weeks. Central Nervous System Infections Cerebrospinal fluid must be cultured as soon as possible after collection. 34 Optimal culturing requires inoculation of blood and chocolate agar plates at the bedside, and laboratory inoculation on the same media from a centrifuged cerebrospinal fluid deposit in order to detect low numbers of organisms. Suitable mycobacterial and fungal media can be inoculated from the cerebrospinal deposit when indicated. Soft Tissue, Intra-Abdominal, and Musculoskeletal Infections Specimens from acute infections require bacterial culture under aerobic and anaerobic conditions to enable the wide range of possible bacterial pathogens to be isolated. In chronic infections, suitable media for the isolation of mycobacteria and fungi should be inoculated and incubated for up to 10 weeks. A variety of tissue cultures can be inoculated to detect viral infections. Gastrointestinal Tract Infections Routine bacterial culture of feces or rectal swabs for salmonella and shigella infections requires MacConkey and desoxycholate-citrate agars and selenite enrichment broth. Specific media can be used to isolate Campylobacter fetus subspecies jejuni, Yersinia enterocolitica, and Vibrio cholerae and V. parahaemolyticus. Detection of enteropathogenic strains of Eschericia coli requires demonstration of enterotoxin production or invasive properties, and these assays are not generally available. Viruses causing diarrhea, such as rotavirus and the Norwalk agent, cannot be cultured at present, and require direct visualization, antigen detection or serology for diagnosis. Genital Tract Infections Gonococci can be isolated by inoculating specimens directly onto Thayer-Martin media or various modifications of this medium. Chlamydia trachomatis can be isolated using tissue culture techniques. Ocular Infections Bacterial, viral, and chlamydial causes of conjunctivitis can be identified using suitable culture techniques for these organisms.
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
IDENTIFICATION OF ORGANISMS ISOLATED
Rapidly growing bacteria, mycobacteria, and fungi are usually identified by their colonial morphology, stained microscopic morphology, and a variety of tests to further differentiate similar organisms using biochemical or antigenic properties of the strain isolated. Most common isolates are readily identified to genus and species in a routine laboratory, but atypical strains, rarely isolated species, and differentiation of phage or serotypes of a strain are referred to laboratories such as State Health Laboratories for identification. Viruses and chlamydia do not grow on cell-free media. Their growth in tissue cultures can be detected in a variety of ways, depending on the organism and its effect on the tissue culture. 25 ANTIMICROBIAL SUSCEPTIBILITY TESTING
One of the most important functions in diagnostic microbiology is the determination of the susceptibility of pathogens to antimicrobial agents. Some organisms are naturally resistant to antimicrobial agents, while others are capable of developing or acquiring resistance. The terms "susceptible" and "resistant" can be used in different contexts-on a clinical basis, these terms refer to the likely response of a patient with a specific infection to a specific antimicrobial agent; on a microbiologic basis, they refer to the ability of an organism to multiply in the presence of a defined concentration of an antimicrobial agent. 5 These terms are at best arbitrary, and are limited by a number of in vivo and in vitro factors. Examples of in vivo factors include host-defense mechanisms, as well as the concentrations of antimicrobial agents achieved at the site of infection, the natural course of the infection, the nature and severity of the infection, delay in initiation of therapy, and effects of other therapeutic measures such as surgical drainage of abscesses. In vitro factors that can affect the susceptibility of organisms include the medium, atmosphere, inoculum size, and length and temperature of incubation. These in vitro factors have been very carefully standardized, so that the outcome of an infection is influenced mainly by in. vivo variations. Organisms are described as susceptible if they are inhibited in vitro by a concentration of an antimicrobial agent that is lower than usual concentrations of that agent in the blood of patients treated with usual doses of the agent. Resistant organisms are either not inhibited, or are inhibited at concentrations above those attainable clinically. In some instances, organisms are regarded as relatively or partially resistant if they are less susceptible to an antimicrobial agent than most other similar isolates. Susceptibility and resistance are expressed as the minimal inhibitory concentration (MIC) of an antimicrobial agent required to inhibit a defined population of organisms under standard conditions, or are based on indirect correlation of a test such as disk diffusion to MICs. Results can either be expressed as MIC values in tJ.g/ml or can be categorized based on achievable levels of antimicrobial agents in various body sites. The meth-
}ACOBS AND CAROLYN MYERS
ods used for determination of susceptibility and their categorical interpretation will be discussed. MIC Determination MICs are determined by incorporating serial two-fold dilutions of antimicrobial agents in a bacteriologic growth medium such as MuellerHinton medium. 27 These dilutions can be in test tubes (macrodilution method), in microdilution wells (microdilution method), or in Petri dishes using media solidified with agar (agar dilution method). For the macroand microdilution methods, inocula of 5 X lOS organisms per ml are used. For the agar dilution method, plates are spot-inoculated with a replicating device delivering inocula containing 1()4 organisms. Tests are incubated at 35°C overnight, and the lowest antimicrobial concentration completely inhibiting the organism is read as the MIC. Determination of minimal bactericidal concentration (MBC) can be performed by subculturing suitable volumes from tubes showing no growth in the macrodilution method-the MBC is read as the lowest concentration producing ~99. 9 per cent reduction in the original inoculum-that is, reduction from 5 X 105/ml to <5 X 1()2/ml. MBCs should not be performed from microdilution or agar dilution tests, as these determinations have not been adequately standardized. The macrodilution MIC method provides reproducible results and can be used to determine MBCs. This method, however, is time-consuming and expensive, and is used only when indicated. Microdilution methods provide excellent results, which are identical to those of macrodilution methods for gram-positive bacteria, and one dilution lower for enteric gram-negative bacilli. 6 Microdilution trays are commercially available either frozen or lyophilized, and can be routinely used by microbiology laboratories. The agar dilution method is probably the most reproducible MIC method, and is also most easily modified to allow for the growth of fastidious organisms. This method also allows for the testing of up to 36 strains simultaneously on a series of plates, and is therefore useful for testing large numbers of organisms. Disadvantages of this method include the need to prepare the plates, as they are not commercially available in smaller laboratories, and the lack of sufficient numbers of strains. Once MIC of various antimicrobial agents for an organism have been determined, they can be expressed as actual values in jl.g/ml or they can be compared to achievable drug levels in various body sites and results interpreted into categories. Interpretation is generally based on achievable blood levels of antimicrobial agents, and a recently proposed interpretation scheme defines four susceptibility categories: 27 Very susceptible--organism is readily inhibited by levels of antimicrobial attained in the blood on usual dosage, including oral when applicable. Moderately susceptible--organism is inhibited only by blood levels achieved with fairly high dosage. Moderately resistant--organism is inhibited by levels achieved where drug is concentrated (for example, in the urine). Very resistant--organism is resistant to usually achievable levels. These categories are broad; final interpretation depends on the pa-
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
tient' s presentation and the pharmacokinetics of drugs being used. The MIC ranges corresponding to each category for various antimicrobial agents is given in Table 2. These interpretations do not apply to sites of poor drug penetration, such as the cerebrospinal fluid, and should not be applied to isolates causing meningitis.
Disk Diffusion Testing This method is based on the inverse relationship between the MIC and the diameter of inhibition around an antimicrobial containing disk of an organism under standard conditions. 28 Antimicrobial-containing disks (up to 12 on a 15-cm plate) are placed on a plate seeded with a specific inoculum of organisms and incubated overnight at 35°G. Zones of inhibition around each disk are measured and interpreted according to a threecategory system as susceptible, intermediate, or resistant based on zonediameter breakpoints (Table 3).28. 29 These breakpoints are based on choice of two MIC breakpoints, and categorical definitions are similar to those given for MICs above. Disk diffusion results, however, are not always as reliable as MIC results, and the intermediate category often is regarded as indeterminant, requiring MIC determination for accurate interpretation.
Table 2. ANTIMICROBIC
Ampicillin* Penicillin Gt Carbenicillin Ticarcillin Methicillin Nafcillin Oxacillin Cephalosporins:f: Amikacin Gentamicin Kanamycin Tobramycin Chloramphenicol Clindamycin Erythromycin Polymyxins Tetracycline Vancomycin Nalidixic Acid§ Nitrofurantoin§ Sulfonamides§ Trimethoprim/ Sulfamethoxazole§
Suggested Guidelines for Interpretation of MICs VERY SUSCEPTIBLE
:s0.25 :S0.03 :S32 :S16
0.5-16 0.06-16 64-128 32-64
32-128 32-128 256 128
4-16 4-16 1-4 4-16 1-4 2-8 1-4 1-4 4-8 2-8 1-16 32 64 64-128
32-256 32-64 8-16 32-64 8-16 16 8-32 8-32 16-Q4 32-64 64-128 128 256-1280
>128 >128 >256 >128 >4 >2 >2 >256 >64 >16 >64 >16 >16 >32 >32 >8 >64 >64 >128 >128 >1280
:s2 :S2 :S2 :s0.5 :S2 :s0.5 :S1 :s0.5 :s0.5 :S2 :S1 :s0.5 :S16 :S32 :S32 :s0.5/9.5
*Includes amoxicillin and bicampicillin. tStaphylococci with penicillin MICs 2::0.06 j.i.g/ml may produce betalactamase. All betalactamase positive staphylococci should be considered resistant to penicillin. :f:Includes first and second generation cephalosporins only. §For use with urinary tract infections.
Zone Diameter Interpretive Standards and Approximate MIG Correlates APPROXIMATE MIG
Amikacinh Ampicillin' when testing gram-negative enteric organisms and enterococci Ampicillin' when testing staphylococcid and penicillin G-susceptible microorganisms Ampicillin' when testing H aemophilus species• Carbenicillin when testing the Enterobacteriaceae Carbenicillin when testing Pseudomonas aeruginosa Cefamandole' Cefotaxime' Cefoxitinl Cephalothin• Chloramphenicol Clindamycinh Colistini Erythromycin Gentamicinh Kanamycin Methicillink Nafcillink Nalidixic Acidi Nitrofurantoin! Oxacillink Penicillin G when testing staphylococcim Penicillin G when testing other microorganisms• Polymyxin Bi Sulfonamidesl.o TetracyclineP Ticarcillin when testing P. aeruginosa Trimethoprimi.o Trimethoprim-sulfamethoxazoleo Tobramycin• Vancomycin
ZONE DIAMETER NEAREST WHOLE MM
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30 f)-g 10 f)-g
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14-16 15--17 15--22 15--17 15--17 13--17 15--16 S--10 14-17 13--14 14-17 10-13 ll-12 14-18 15--16 ll-12 21-28 12-21 S--11 13--16 15--18 12-14 ll-15 ll-15 13--14 10-ll
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"'=256 "'= 32 "'= 64 "'= 32 "'= 32 "'= 25 2 4 8 8 "'= 25 "'= 16 8 "'= 32 "'=100 8
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"'= 32 f1g/ml "'= 50 units/ml "'=350 f1g/ml "'= 12 f1g/ml "'=128 f1g/ml "'= 16 f1g/ml "'=81152 f1g/ml 8 f1g/ml
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f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml j f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml j f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml f1g/ml
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z> tl (')
a. These correlates are not meant for use as breakpoints for susceptibility categorization with dilution MIC tests as described in Table 2. b. The zone sizes obtained with aminoglycosides, particularly when testing Pseudomonas aeruginosa, are very medium dependent because of variations in divalent cation content. The zone diameter interpretive standards for amikacin, gentamicin, and tobramycin are to be used only with Mueller-Hinton
medium that has yielded zone diameters within the correct range when tested with P. aeruginosa ATCC 27853. In addition, the amikacin disk must be 30 ~~og rather than the 10 !Jog disk used previously. Organisms in the intermediate category may be either susceptible or resistant when tested by dilution methods and should therefore more properly be classified as "indeterminate" in their susceptibility to aminoglycosides. c. Class disk for ampicillin, amoxicillin, cyclacillin, and hetacillin. d. Resistant strains of S. aureus produce 13-lactamase and the use of the 10 unit penicillin disk is preferred. e. For testing Haemophilus use Mueller-Hinton agar supplemented with 1 per cent hemoglobin and 1 per cent IsoVitaleX (BBL), Supplement VX (Difco) or an equivalent synthetic supplement. Adjust pH to 7.2. Prepare the inoculum by suspending growth from a 24-hour chocolate agar plate in Mueller-Hinton broth to the density of a turbidity standard. The vast majority of ampicillin-resistant strains of Haemophilus produce detectable 13-lactamase. f. Cefamandole, cefoxitin and cefotaxime are recently released cephalosporins having a wider spectrum of activity against gram-negative bacilli than do other previously approved cephalosporins. Therefore, the cephalothin disk cannot be used as the class disk for these three drugs. g. The cephalothin disk is used for testing susceptibility to cephalothin, cefaclor, cefadroxil, cefazolin, cephalexin, cephaloridine, cephapirin, and cephradine. Cefamandole, cefoxitin and cefotaxime must be tested separately. Staphylococcus aureus exhibiting resistance to methicillin, nafcillin or oxacillin disks should be reported as resistant to cephalosporin-type antimicrobics, regardless of zone diameter, because in most cases infections caused by these organisms are clinically resistant to cephalosporins. Methicillin-resistant S. epidermidis infections also may not respond to cephalosporins. h. The clindamycin disk is used for testing susceptibility to both clindamycin and lincomycin. j. Colistin and polymyxin B diffuse poorly in agar, and the diffusion method is thus less accurate than with other antimicrobics. Resistance is always significant, but when treatment of systemic infections caused by susceptible strains is being considered, results of a diffusion test should be confirmed with those of a dilution method. MIC correlates cannot be calculated reliably from regression analysis. k. Of the antistaphylococcal 13-lactamase resistant penicillins, either oxacillin, nafcillin, or methicillin may be tested, and results can be applied to the other two of these drugs and to cloxacillin and dicloxacillin. Oxacillin and nafcillin are more resistant to degradation in storage. Cloxacillin disks should not be used because they may not detect methicillin-resistant S. aureus. When an intermediate result is obtained with S. aureus, the strains should be further investigated to determine if they are heteroresistant. I. Susceptibility data for nalidixic acid, nitrofurantoin, sulfonamides, and trimethoprim apply only to organisms isolated from urinary-tract infections. m. Penicillin G should be used to test the susceptibility of all penicillinase-sensitive penicillins, such as ampicillin, amoxicillin, hetacillin, carbenicillin and ticarcillin. Results may also be applied to phenoxymethyl penicillin or phenethicillin. The intermediate category usually contains penicillinaseproducing isolates and should be considered resistant to therapy. n. Intermediate category includes some microorganisms, such as enterococci, and certain gram-negative bacilli that may cause systemic infections treatable with high parenteral dosages of benzyl penicillin but not of orally administered phenoxymethyl penicillin or phenethicillin. For pneumococci and gonococci refer to special interpretations. o. The 250 or 300 ~~og sulfisoxazole disks can be used for any of the commercially available sulfonamides. Blood-containing media, except media containing lysed horse blood, are not satisfactory for testing sulfonamides. The Mueller-Hinton agar should be as thymidine-free as possible for sulfonamide and/or trimethoprim testing. p. Tetracycline is the class disc for all tetracyclines, and the results can be applied to chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, and oxytetracycline. However, some in vitro data show that certain organisms may be more susceptible to doxycycline and minocycline than to tetracycline. q. The category "intermediate" should be reported. Infections with bacteria of "intermediate" susceptibility may be considered moderately susceptible and may respond to antimicrobial agents with a wide safe dosage range.
}ACOBS AND CAROLYN MYERS
Nevertheless, disk diffusion results using the "Kirby-Bauer" procedure produce good results when used as described. Limitations of disk diffusion include the range of drugs and organisms that can be tested~mly enteric gram-negative bacilli, staphylococci, and enterococci can be tested on the standard Mueller-Hinton medium; pneumococci can be tested on this medium supplemented with 5 per cent sheep blood, and Haemophilus can be tested on the same medium supplemented with 1 per cent hemoglobin and l per cent IsoVitaleX. Anaerobes cannot be tested by disk diffusion. Recommended methods for susceptibility testing of various common isolates are given in Table 4, and recommended antimicrobial agents to be tested for each group of organisms are given in Table 5. Rapid Susceptibility Testing Methods The obvious need for results of antimicrobial susceptibility of pathogens to be available as soon as possible has been addressed by three commercial systems. These systems, which are automated to a varying degree, can produce susceptibility results in 3 to 7 hours, instead of 18 to 24 hours using conventional techniques. 33 Organisms that can be tested in these systems include staphylococci, enterococci, and enteric gram-negative bacilli. The three systems available are the Autobac (General Diagnostics), MS-2 (Abbott Laboratories), and AutoMicrobic System (Vitek Systems). 20 These systems can test 9 to 13 antimicrobial agents at a time, and for two of the three systems agents to be tested can be selected by the user. Agents producing most errors in these systems were ampicillin, tetracycline and nitrofurantoin, particularly when test strains were Klebsiella, Providencia, Serratia, and Citrobacter species. These systems therefore must be used with caution, and results should be confirmed using an MIC method. Another limitation of these systems is the lag between the introduction of new drugs and the availability of these for testing in the system. Table 4.
Aerobes Gram-positive isolates Staphylococci Enterococci Pneumococci Other streptococci Gram negative isolates Enteric bacilli* Haemophilus Otherst Anaerobes
Methods for Susceptibility Testing of Bacteria MEDIUM FOR MIC DETERMINATIONt
MEDIUM FOR DISK DIFFUSION TESTING
Mueller-Hinton Mueller-Hinton Mueller-Hinton+5% blood Mueller-Hinton+5% blood
Mueller-Hinton Mueller-Hinton Mueller-Hinton+5% blood
Mueller-Hinton Mueller-Hinton+ 1% Hb + 1% IsoVitaleX Various§ Wilkins-Chalgren
Mueller-Hinton Mueller-Hinton+ 1% Hb + 1% IsoVitalex NA NA
*Including enterobacteriaceae and Pseudomonas aeruginosa. tlncluding various nonfermenting bacilli and fastidious aerobic cocci and bacilli. tUsing macrodilution, microdilution, or agar incorporation techniques. §A variety of media may be used to support the growth of fastidious organisms. !INA, not applicable.
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
Recommended Antimicrobial Agents to be Tested According to Organism and Site of Isolation'
Enteric gram-negative isolates from urine Ampicillinb Sulfisoxazoleb Trimethoprim Tetracyclineb Nitrofurantoin Carbenicillin' Nalidixic acid
Gentamicin Tobramycin Amikacin Cephalothinb. d Cefoxitind Cefamandoled
Enteric gram-negative isolates from other sites Ampicillinb Tetracyclineb Trimethoprim Chloramphenicol Carbenicillin' Gentamicin
Tobramycin Amikacin Cephalothin h. d Cefoxitind Cefamandoled
Penicillin G Oxacillin or methicillin Cephalothin Erythromycin Clindamycin
Vancomycin Gentamicin Tetracycline Nitrofurantoin'
Ampicillinb Penicillin G Erythromycin Tetracyclineb
Chloramphenicol Trimethoprim' Nitrofurantoin'
Streptococcus pneumoniae Penicillin G1 Chloramphenicol
Haemophilus influenzae Ampicillinb Chloramphenicol Tetracyclineb
Erythromycin Trimethoprim Sulfisoxazoleb
Penicillin G Carbenicillin Cefoxitin Chloramphenicol
Clindamycin Tetracyclineb Metronidazole
•Antimicrobial agents given are representative of currently recommended agents. Local patterns of susceptibility may necessitate use of other combinations. The position of new !3-lactam agents has not been finalized, and these agents have not been considered for primary testing. bThese agents represent similar agents, e. g. ampicillin represents amoxicillin, cephalothin represents cephaloridine, cephalexin, cefazoline, cephradine, etc. •Carbenicillin-resistant enterics may be susceptible to mezlocillin and piperacillin. dEnterics resistant to first- and second-generation cephalosporins may be susceptible to third generation cephalosporins. •For urinary isolates only. 'For disk diffusion testing, oxacillin or methicillin disks reflect penicillin G susceptibility more accurately than penicillin G disks.
R. jACOBS AND CAROLYN MYERS
Detection of ~-lactamase Production Most penicillin- and ampicillin-resistant strains of Haemophilus influenzae, Neisseria meningitidis and Staphylococcus aureus produce ~-lactamases, and these can be rapidly demonstrated directly from colonies growing on solid media or on organisms growing in blood culture media. 2 ~-Lactamase production by S. aureus is inducible and may be detected only after growth of strains in the presence of methicillin or oxacillin. Rapid methods suitable for detection of ~-lactamase in H. influenzae include the acidimetric method, which detects a pH change to acid from the production of penicilloic acid from penicillin; the iodometric method, which is based on penicilloic acid reacting with iodine and preventing the iodine from reacting with starch to produce a purple color; and the chromogenic cephalosporin method, in which ~-lactamases cause these substrates-for example, nitrocefin and PADAC-to change color. Reagents for these tests are commercially available and all work well with isolates of H. influenzae, with test results usually able to be interpreted within 10 minutes. TOLERANCE OF STAPHYLOCOCCUS AUREUS TO ~-LACTAM AGENTS Tolerance, the ability of an organism to survive the effects of an antimicrobial agent, has recently been described in S. au reus .46 These strains have low MICs but high MBCs, with the ratio of MBC to MIC of >32 being required to call strains tolerant. Analysis of tolerant strains has shown that a small subpopulation of a strain does not autolyze due to excess of an autolysin inhibitor. The clinical implications of tolerance are as yet undetermined. Up to 50 per cent of strains of S. aureus are tolerant to ~-lactamase-resistant penicillins such as oxacillin and nafcillin, and testing for tolerance by MIC and MBC determination has been suggested for strains responsible for serious staphylococcal infections. ACTIVITY OF ANTIMICROBIAL AGENTS IN COMBINATION As combinations of antimicrobial agents are often used, their interactions are important and can be evaluated in vitro. 2 Combinations are synergistic when their combined effect is greater than the sum of the effects of each, additive when their combined activity is equal to the sum of their effects, indifferent if the combination is only as active as one of the components, or antagonistic when the combination is less active than either drug alone. These effects can be detected by testing the rate of killing of a strain with drugs alone and in combination or by testing multiple drug concentrations and determining MICs and MBCs on these combinations. Combinations are synergistic when concentrations in combination are bactericidal at lower concentrations than either drug alone, and antagonistic when higher.
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
Determination of synergism or antagonism is time-consuming and expensive, and application of such in vitro results to patients has not been adequately defined. Many drug combinations are known to be synergistic in vivo--for example, trimethoprim with sulfonamides; penicillins with aminoglycosides against streptococci and staphylococci; and ticarcillin with aminoglycosides against enteric gram-negative bacilli. 21 Combinations known to be antagonistic generally include a bacteriostatic with a bactericidal agent-for example, tetracycline with penicillin G in pneumococcal meningitis. Combination of chloramphenicol with a ~-lactam agent may also be antagonistic, particularly against enteric gram-negative bacilli causing meningitis.
DETERMINATION OF BACTERICIDAL ACTIVITY IN BODY FLUIDS As a guide to the adequate selection of antimicrobial agents and use of optimal dosage, the bactericidal activity of serum or other body fluids can be determined against the actual pathogen isolated from the patient. 2 Peak and trough serum specimens are collected in conjunction with the administration of antimicrobial agents. Peak levels are drawn 15 to 30 minutes after IV infusion, and 1 hour after IM administration. Trough levels are drawn just before the next drug dose is due. The test, often referred to as the Schlichter test, is performed by preparing serial two-fold dilutions of serum or other body fluid to be tested in Mueller-Hinton broth or pooled normal human serum in 0.5 ml volumes, to which is added 0.5 ml of the patient's organism suspension in MuellerHinton broth to produce a final inoculum of 105 to 1()6 organisms per ml. After overnight incubation, bacteriostatic levels are read (if sera are not too cloudy to obscure this) and tubes subcultured to detect bactericidal levels (>99.9 per cent kill). Results are expressed as serum or other fluid dilutions at which a bacteriostatic and bactericidal effect was detected. Therapy is generally considered adequate if the peak bactericidal level is 1:8 in endocarditis; interpretation in other infections has not been adequately documented. Limitations of this technique are the lack of adequate criteria for interpretation and the inability of some slow-growing or fastidious pathogens to grow under the test conditions used. Determination of the actual level of antimicrobial agents is often more useful, and is discussed under therapeutic drug monitoring.
THERAPEUTIC DRUG MONITOJUNG As described above, it is essential to obtain serum antibiotic concentrations in excess of the in vitro MIC against invading organisms. The relation of dose to serum concentration is well established. with many antimicrobial agents; most do not produce dose-related adverse effects, and monitoring of serum levels is not considered necessary. However, the
JACOBS AND CAROLYN MYERS
aminoglycosides and chloramphenicol are exceptions and do need to be closely monitored. Although "dose-related toxicity" is a feature of chloramphenicol and aminoglycoside usage, there is better correlation of toxicity with serum drug concentration. 38• 39 Wide variability in the correlation of dose to serum concentration is a common finding, being influenced by factors such as body composition, tissue uptake and release of drug, hematocrit, age, sex, liver and kidney function, and disease status. 30 Toxicity A major risk factor (not necessarily causative) for aminoglycoside nephrotoxicity is a pre-dose (trough) serum concentration of 2:2 !J.g/ml for gentamicin and tobramycin and 2:10 !J.g/ml for amikacin. 7, 39 The risk factors for gentamicin ototoxicity in children include use of diuretics, renal insufficiency, and peak serum levels of 2:12 iJ.g/mJ.9 Of the three types of toxicity associated with chloramphenicol, two are dose related. High doses of 2:100 mg/kg/day, particularly in newborns, may precipitate the well-known gray syndrome. 23 Also, a reversible doserelated hematopoietic toxicity generally occurs with doses greater than 50 mg/kg and specifically at chloramphenicol serum concentrations above 25 !J.g/ml, 38 although some data suggest a lower serum concentration cutoff of 10 !J.g/ml. 18• 40 An increase in serum iron of over 120 !J.g/100 ml and TIBC of 50 to 100 per cent are the earliest indicators of this toxicity. 40 Chronic treatment in cystic fibrosis patients has also produced a dose-related optic neuritis.l3 A rarely occurring nonreversible hypoplasia of the bone marrow with severe pancytopenia appears weeks or months after chloramphenicol has been withdrawn. No correlation with the extent of chloramphenicol dosing has been found. 26
DRUG BIODISPOSITION In order to effectively interpret serum antimicrobial concentrations, the principles and assumptions that are used to interpret this information are discussed. Pharmacokinetics The concept of drug biodisposition includes the absorption, distribution, metabolism, and elimination of a drug. Each has an effect on the concentration of drug in the blood. The basic pharmacokinetic study that describes changes in drug concentration over time must take into account these many effects. The standard model used for such a study proposes that the patient's condition does not change and that the drug distributes evenly into one basic compartment, the volume of distribution (V 0 ). After distribution, serum drug diminishes through elimination according to firstorder or zero-order kinetics. In first-order kinetics the process of elimination is exponential, which means that half of the drug present at any time will be eliminated following a constant time interval called the half-life (t112). Zero-order kinetics are linear and encountered only when the process
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
of elimination is saturated so that only a fixed amount of drug can be eliminated within a given time period. Half-life, which is expressed in units of time, also translates through the exponential curve into a rate (13) with the units of 1/time where 13 = 0. 693/t 112 • The absorption phase may also be characterized by a rate (a). The mathematical expression for the excretion of drug from the volume it occupies in the body is total body clearance (C~8). It has the units of volume/time and ClTB = I3V 0 . Both the V0 and 13 can be determined by plotting actual serum concentrations of drug at varying times on semilog paper. Early time points represent absorption and distribution of drug. The half-life can be determined directly from the slope of the later, linear elimination phase. Extrapolation of this phase back to time zero gives the initial theoretical serum concentration (C0), which may be divided into the dose to obtain V0 ( D~se = v 0 ). 0
Patient Age Pharmacokinetics in neonates are very different from those in other age groups. The concentration of serum proteins is 80 per cent of the adult concentration and the enzyme systems responsible for glucuoronidation are not mature at birth. Both of these factors increase the effective serum chloramphenicol concentration. Glomerular filtration doubles within the first two weeks alone and increases sevenfold between birth and one year. Is This will affect the elimination of both chloramphenicol and the aminoglycosides. All these factors contribute to the exceedingly long half-life of chloramphenicol at birth (as long as three days). Within a few days of life this drops to one day and by two weeks is down to 10 hours. Finally, between one and four months the adult half-life of four to five hours is achieved. 12• 43 A similar phenomenon occurs with the aminoglycosides. The half-life decreases by almost one-half during the first week of life. 17 Adult values of two hours are observed in infants, while teenagers show values of less than two hours.l6 Dosage Drugs are usually administered to maintain a minimum serum concentration and to avoid extreme concentration fluctuation, so it is common to dose every half-life. When the amount of drug administered equals the amount of drug eliminated over a dosing interval, drug biodisposition has reached a steady state. This cannot occur after a single dose, since only 50 per cent of the dose can be eliminated after the one half-life interval; therefore drug will accumulate over the first four or five doses. To bypass this accumulation an initial loading dose can be given. Changes in a patient's condition can change pharmacokinetics, so drug concentrations achieved with new dosage regimens should be checked after four or five doses, when a new steady state has been established. If the drug is to be used longer than the typical 10-day period, concentrations should also be checked periodically. For example, peak and trough aminoglycoside levels will slowly increase due to tissue accumulation. 7, 37
JACOBS AND CAROLYN MYERS
APPROPRIATE SERUM SAMPLING After maintenance dosing has commenced, it is then necessary to confirm the anticipated antibiotic concentration in serum.
Timing Aminoglycoside peak and trough concentrations should be determined, since high peaks and high troughs have been correlated with ototoxicity and nephrotoxicity respectively. Peaks are drawn 30 minutes after the end of a 30-minute infusion or 60 minutes after an intramuscular injection. For pharmacokinetic evaluation two intermediate samples within four hours after infusion or injection should be drawn. For chloramphenicol, the peak, as one indicator of toxicity, should be drawn one hour after the end of an intravenous infusion or two hours after an oral dose. For pharmacokinetic evaluation a trough and one or two intermediate samples should be drawn as well.
Interpretation It is extremely important to obtain the following information in order to draw any useful conclusions from serum antimicrobial levels: (l) the time the sample is drawn, (2) drug type, (3) time of most recent dose, (4) dosage, (5) route of administration, (6) dose frequency, (7) administration of other drugs, and (8) details of patient's age, weight, and clinical condition.
ROLE OF THE LABORATORY IN DRUG MONITORING Methods of drug analysis now allow analysis to be performed on the contents of 3 to 5 hematocrit tubes. Special mention needs to be made of interactions of the aminoglycosides with ~-lactams.l 4 , 32 At 37°C and in concentrations of 10 ~J.og/ml, gentamicin in serum shows no significant loss in activity over 48 hours in the presence of 50 !J.og/ml ampicillin, carbenicillin, cloxacillin, methicillin, benzylpenicillin, or ticarcillin. However, carbenicillin at 200 IJ.og/ml causes a 60 per cent loss in gentamicin activity over the same time period. 32 This loss of activity is both time dependent and concentration dependent. 14· 32 Tobramycin is more sensitive to inactivation than gentamicin and netilmicin less sensitive. Amikacin is the most stable aminoglycoside. It has also been shown that rates of inactivation are slower at lower temperatures, and freezing samples at - 70°C halts inactivation. Therefore, blood specimens for aminoglycoside analysis should be refrigerated or centrifuged and frozen as soon as possible after collection. Chloramphenicol, on the other hand, is very stable, and serum samples can be held or transported at ambient temperatures. 31
Methods Drugs can be assayed using microbiologic, biochemical, and chromatographic methods. Microbiologic methods are versatile and economical but usually require 24 hours for their performance; specificity is also a problem for patients receiving multiple antimicrobial agents.
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
Radioimmunoassay (RIA), enzyme radioassay (ERA), and fluoroimmunoassay (FIA) are preferentially run in batches. Up to 30 assays can be performed within three hours. The coefficients of variation are best for the ERA (seldom exceeding 7 per cent) and poorest for the RIA (often greater than 10 per cent). However, while the limit of detection is between 0.3 and 1.0 J.Lg/ml for the ERA and FIA assays, the RIA can detect concentrations down to 0.03 J.Lg/ml. The above assays require no more than 10 J.Ll of sample. These methods require an initial investment in a counter or fluorometer. Gentamicin, tobramycin, and amikacin kits are available for the RIA, ERA, and FIA methodologies [Diagnostics Products Corp.; Miles Laboratories, Inc.; P.L. Biochemicals]. The EMIT method [Syva Co.], which employs unique sets of enzyme and antibody reagents, can be used to assay 50 J.Ll samples containing gentamicin, tobramycin, and amikacin with a precision within 10 per cent. This method is being developed for chloramphenicol. The only equipment required is a spectrophotometer, pipetter-diluter, and printer, but reagents are expensive. Since assays are performed individually within a few minutes, the method is equally useful with both small and large workloads. Both high pressure liquid chromatography and gas chromatography with electron capture require a large investment in equipment, but both systems can be adapted to a variety of drug assays. In general, sample chromatography takes no more than 10 minutes, but sample preparation, instrument set-up, and chromatography of standards can take 30 to 90 minutes. Chloramphenicol is more amenable to chromatography than the aminoglycosides, and does not require derivation in the case of high pressure liquid chromatography. Coefficients of variation for chromatography lie between 5 and 10 per cent, and 0.05 ml samples allow a limit of detection of about 1 J.Lg/ml. A latex agglutination inhibition card test for gentamicin has been developed recently. 8 While this method is very simple and rapid (12 minutes) and requires no instrumentation, the precision varies widely, sometimes exceeding 20 per cent. Caution in the use and interpretation of this method should be used.
SEROLOGIC DIAGNOSIS OF INFECTIONS The detection of antibodies to infecting organisms or their products can be of vital importance in confirming a clinical diagnosis. Advantages of serology over cultural methods include disease where cultures are difficult to obtain or evaluate (for example, Mycoplasma pneumoniae infection, toxoplasmosis, Legionnaires' disease), where organisms are difficult or hazardous to isolate (for example, tularemia, syphilis, rickettsial infections, hepatitis viruses) or where antimicrobial therapy was initiated before culture were obtained (for example, typhoid fever, group A streptococcal pharyngitis). 44 Disadvantages associated with the serologic diagnosis of infections include the need to collect acute and convalescent phase sera 7 to 10 days or more apart, the need to test for antibodies to a wide variety of organisms responsible for infections such as viral respiratory tract infections, cross-
R. JACOBS AND CAROLYN MYERS
reactions, the failure of some patients to produce a serologic response with proven infection, and the obvious delay before results are available to guide therapy. A wide variety of techniques is available for detection of antibodies, including precipitin tests, immunodiffusion, whole-organism agglutination, agglutination of antigen-coated latex particles or red cells, radio- and enzyme-linked immunoassay (RIA and ELISA), hemagglutination inhibition, indirect fluorescent antibody (IFA) test, viral neutralization, and complement fixation tests (CFT). Many of these tests are difficult to perform, but commercial reagents are becoming increasingly available, and newer techniques, such as ELISA, provide high levels of sensitivity and specificity for antibody detection. In addition, use of IgG and IgM specific conjugates can separate the antibody response into classes using RIA, IFA, and ELISA methods and can provide information on the presence of a recent and congenital infection if IgM-specific antibodies are detected. Examples of specific serologic tests are given below. Bacterial Infections Serology is useful in the diagnosis of enteric fever, brucellosis, and tularemia by agglutination testing; Legionnaires' disease by IFA; syphilis by VORL, RPR, and fluorescent treponemal antibody methods; group A streptococcal infections by detection of antibodies against streptolysin 0, DNase, and other antigens; and staphylococcal infections by detection of teichoic acid antibodies. Viral Infections A large number of viral infections can be diagnosed serologically, including herpesviruses, adenovirus, respiratory viruses (influenza, parainfluenza, respiratory syncytial, and so on), arboviruses causing encephalitis, enteroviruses (polio, coxsackie), measles, mumps, and rubella. TechniqueSused include CFT, IFA, ELISA, hemagglutination inhibition, and neutralization tests. When serologic tests are required, the laboratory must be informed of the patient's clinical presentation and against which viral agents antibodies should be sought. Many of these investigations are available only in reference laboratories, and test results are often delayed. Fungal Infections Serologic tests are useful in the diagnosis of histoplasmosis, coccidiodomycosis, blastomycosis, and some forms of aspergillosis. CFT and precipitin tests are usually used, and results are often most useful in patients living in nonendemic areas who have recently traveled to an endemic area of histoplasmosis or coccidiodomycosis. Rickettsial Infections Non-specific agglutination tests (Weil-Felix test) have been used to diagnose typhus and spotted fever, and specific complement-fixation tests are available at reference laboratories to detect antibodies to these and other rickettsial infections such as Q-fever.
DIAGNOSTIC MICROBIOLOGY AND THERAPEUTIC DRUG MONITORING
Parasitic Infections Antibodies can be detected against a wide variety of parasites. Examples include toxoplasmosis (IRA and ELISA) and amebiasis (latex agglutination). Congenital Infections Infections transmitted in utero or during delivery can be caused by syphilis, rubella virus, Toxoplasma gondii, herpes simplex virus, and cytomegalovirus, and these infections are often best detected serologically. As maternal lgG, but not lgM, crosses the placenta, detection of lgMspecific antibody, or persistence or titer rise of a serologic test during the first few months of life, is required for a congenital infection to be diagnosed serologically.
EPIDEMIOLOGY While identification of an isolate to species or even subspecies level, and determination of antimicrobial susceptibility to a battery of agents, may not always be of direct benefit to the patient from whom the specimen was derived, such information can be of major epidemiologic importance. 24 Epidemics can be detected and monitored by following the isolation of a species, biotype or serotype of an organism, which often has a distinctive antimicrobial susceptibility pattern. Use of common methods and interpretative criteria can be invaluable in tracing contaminated infusion fluids, differentiating species of yeasts, differentiating enterococci from Streptococcus bovis, and in monitoring nosocomial infections. 24 Excessive detail in identification of organisms can be confusing because of the large number of organisms found in human infections and the rapid changes in nomenclature that have occurred. However, without adequate laboratory testing, recognition of nonsocomial outbreaks, unusual organisms, or resistant strains of organisms that are usually susceptible will be delayed or entirely missed.
REFERENCES 1. Anhalt, J. P, Kenny, G. E., and Rytel, M. W.: Cumitech 8, Detection of microbial antigens by counterimmunoelectrophoresis. Gavan, T. L. (ed.). American Society for Microbiology, Washington, D.C., 1978. 2. Anhalt, J. P., Sabath, L. D., and Barry, A. L. Special tests: Batericidal activity, activity of antimicrobics in combination, and detection of 13-lactamase production. In Lennette, E. H., Balows, A., Hausler, J. R., et al. (eds.): Manual of Clinical Microbiology. American Society for Microbiology. 3rd ed. Washington, D.C., 1980, p. 478. 3. Assael, B. M., Cavanna, G., Jusko, W. J., et al.: Multiexponential elimination of gentamicin: A kinetic study during development. Dev. Pharmacol. Ther., I:171, 1980. 4. Barrett, F. F.: Use of the bacteriology laboratory. In Feigin, R. D., and Cherry, J. D. (eds.): Textbook of Pediatric Infectious Diseases Philadelphia, W. B. Saunders Co., 1981, p. 1797. 5. Barry, A. L.: The Antimicrobial Susceptibility Test: Principles and Practices. Philadelphia, Lea and Febiger, 1976.
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