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Direct detection of infections using gas liquid chromatography
References 1. Anderson, E. T., L. S. Young, and
W. L. Hewltl. 1978. Antimicrobial synergism in the therapy of gramnegative rod bacteremia. Chemotherapy 24:45-54. 2. Berenbaum, M. C. 1978. A method for testing for synergy with any number of agents. J. Infect. Dis. 137: 122-130. 3. Klastersky, J., R. Cappel, and D. Danean, 1972, Clinical significance of in vitro synergism between antibiotics in gram-negative infections. Antimicrob. Agents Chemother. 2:470-475. 4. KIastersky, J., C. Hensgens, and F, Meunier-Carpentier, 1976, Comparative effectiveness of combinations of amikacin with penicillin G and amikacin with carbenicillin in
5.
6. 7.
8.
gram-negative septicemia: Doubleblind clinical trial. J. Infect. Dis. 134(Suppl.):S433-S440. Klaslersky, J., F. Meunier-Carpentier, and J. Prevost. 1977. Significance of antimicrobial synergism for the outcome of gram-negative sepsis. Am. J. Med. Sci. 273:157-167. McCabe, W. R., and G. G. Jackson. 1965. Treatment of pyeIonephritis. N. Engl. J, Med. 272:1037-1044. Moellering, R. C., Jr., C. Wennersten, and A. N. Weinberg. 1971. Studies on antibiotic synergism against enterococci. I. Bacteriologic studies. J. Lab. Clin. Med, 77: 821-828. Norden, C. W., H. Wentzel, and E, Keleti. I979. Comparison of techniques for measurement of in vitro antibiotic synergism. J. Infect. Dis. 140:629-633.
9. Reyes, M. P., M. R. EI-Khatib, W..t. Brown, F. Smith, and A. M. Lerner. 1979. Synergy between
carbenicillin and an aminoglycoside (gentamicin or tobramycin) against Pseudomonas aeruginosa isolated from patients with endocarditis and sensitivity of isolates to normal human serum, J. Infect. Dis, 140: 192-202. 10. Young, L. S. 1978. Review of clinical significance of synergy in gramnegative infections at the University of California Los Angeles Hospital. Infection 6(Suppl. 1~:$47-$52
Editorial
Direct Detection of Infections Using Gas Liquid Chromatography
Timothy E. Kiehn, Ph.D. Diagnostic Microbiology Laboratory Memorial Hospital New York, New York 10021 The direct analysis by gas-liquid chromatograph]/(GLC) of tissue homogenates, pus, and body fluids offers the possibility for a more rapid diagnosis of infectious diseases. GLC techniques were first described in the 1940s, and significant advances in techniques and instrumentation have occurred since. Initially, gas chromatography procedures were used primarily by the analytical chemist. The range of compounds that can be analyzed was greatly broadened by the development of procedures for the formation of volatile derivatives. GLC allows separation, quantitation, and tentative identification of many compounds of biological interest. In the 1960s microbiologists first utilized this capability in the identification of fermentation products and as an aid in bacterial classification. Multicomponent analysis of bacterial byproducts is now a routine procedure used as an aid in anaerobe identification. These techniques, however, ii
require that the organism first be isolated and cultured before the cells or the media are analyzed by GLC. The more desirable procedure of directly analyzing patient fluids or tissues for detection and identification of infectious agents was first studied in the late 1960s. Progress in this area has been less rapid due to the complexity of clinical material. In the ensuing 10 years, further advances in instrumentation and techniques, and a series of subsequent diagnostic successes have been made. A variety of column materials, derivatives, and detection methods are now available. Reliable and inexpensive instruments with thermal conductivity detectors are commonly used in the anaerobic laboratory and for the determination of lactic acid in cerebrospinal fluid (CSF). More sensitive flame-ionization and electron-capture detectors are usually necessary for more complex examinations. Fluids may be examined for the presence of single compounds, or an analysis of multiple compounds or "peaks" may be conducted. When examining complex chromatograms (fingerprint analysis) it is helpful to use an integrator for the evaluation of retention times and peak areas. Also, the GLC may be linked to a computer for collation and interpretation of the
chromatograms. Automatic samplers are also available, which allow analysis to continue after working hours. More specific identification of the compounds isolated can be achieved with the use o f a mass spectrometer. Direct tissue or body fluid analysis has been directed primarily at the diagnosis of respiratory infections, meningitis, arthritis, and septicemia. Knowledge gained in anaerobe identification has been utilized in the direct analysis of serous fluid or pus for the detection of fatty acids produced by anaerobic bacteria (4). These acids, which are products of bacterial metabolism, are present in increased quantities distinct from normal or background levels. For example, increased amounts of succinic acid have been found in clinical material from infections by Bacteroides fragilis. The early detection of this penicillin-resistant anaerobe is of clinical importance. GLC has been used to detect increased levels of lactic acid in CSF (1). Levels of this acid are increased during bacterial but not viral meningitis. The technique is also useful in monitoring therapy because the lactic acid level returns to normal as the organism is cleared. Tuberculous, cryptococcal, viral (3), and bacterial
(6) meningitis have also been differentiated by comparison of the CSF chromatographic pattern, sometimes with computer assistance. Bacteria commonly causing septic arthritis, such as staphylococci, streptococci, and neisseriae, have been directly identified in synovial fluids by electron-capture GLC (2). There have been few reports of the direct diagnosis of septicemia by GLC (7). This is due to the complexity of the matrix, the sensitivi.ty of the analysis required, and the difficulty of assembling well-characterized patient groups. Complex pattern comparison has met with little success; a more realistic approach may be metabolite detection, such as the increased arabinitol levels we have reported in invasive candidiasis (5). What are the advantages and difficulties associated with the use of GLC in the diagnostic microbiology laboratory? There are several important advantages to this procedure. It is rapid; most analyses of body fluids or tissues can be performed within one to two hours. Cultures take at least one day, and gram stains, although rapid, are not always helpful. GLC results are usually not affected by recent prior antimicrobial therapy, while culture growth may be inhibited by the antibiotic. GLC may be used to monitor therapy because microbial metabolites may disappear or return to normal levels with appropriate therapy. Other rapid diagnostic procedures have limitations. Immunologic techniques that
rely on detection of antibody may not provide early diagnosis or may not be helpful in immunocompromised patients. Countercurrent immunoelectrophoresis requires high-titered specific antisera, and reports indicate a 10-20% false-negative rate. The limulus assay for the diagnosis of bacterial meningitis is restricted to gram-negative organisms. Disadvantages or problems with GLC must also be considered. Initial cost of the equipment is certainly a factor. In some cases, as with lactic acid levels in meningitis, available enzyme assays are as reliable as GLC and do not require the use of expensive equipment. Training of personnel requires considerable time. The infecting organism must produce compounds that are detectable by the procedures employed. For example, not all strains of Bacteroides produce succinie acid. Much of the research thus far has been performed by only a few groups of investigators. Other laboratories must contribute information so that the reliability of these procedures may be determined. The use of GLC for the direct detection of infections is a promising field. However, significant advances in the use of GLC and other analytic techniques such as high-pressure liquid chromatography will only be made when we increase our collaboration with our colleagues in other technical disciplines. For example, our laboratory's success with GLC in the detection of Candida metabolites didn't begin until we started talking to competent biochemists.
References 1. Brook, I., K. S. Bricknell, G. D. Overturf, and S. M. Finegold. 1978. Measurement of lactic acid in cerebrospinal fluid of patients with infections of the central nervous system. J. Infect. Dis. 137:384-390. 2. Brooks, J. B., D. S. Kellog, C. C. Alley, H. B. Short, H. H. Handsfield, and B. Huff. 1974. Gas chromatography as a potential means of diagnosing arthritis. I. Differentiation between staphylococcal, streptococcal, gonococcal, and traumatic arthritis. J. Infect. Dis. 129:660-668. 3. Craven, R. B., J. B. Brooks, D. C. Edman, J. D. Converse, J. Greenlee, D. Schlossberg, T. Furlow, J. M. Gwaltney, Jr., and W. F. Miner. 1977. Rapid diagnosis of lymphocytic meningitis by frequency-pulsed electron capture gas-liquid chromatography: Differentiation of tuberculous, cryptococcal, and viral meningitis. J. Clin. Microbiol. 6:27-32. 4. Gorbach, S. L., J. W. Mayhew, J. G. Bartlett, H. Thadepalli, and A. B. Onderdonk. 1976. Rapid diagnosis of anaerobic infections by direct gasliquid chromatography of clinical specimens. J. Clin. Invest. 57:478-484. 5. Kiehn, T. E., E. M. Bernard, J. W. M. Gold, and D. Armstrong. 1979. Candidiasis: Detection by gas-liquid chromatography of D-arabinitol, a fungal metabolite, in human serum. Science 206:577-580. 6. La Force, F. M., J. L. Brice, and T. G. Tornabene. 1979. Diagnosis of bacterial meningitis by gas-liquid chromatography. II. Analysis of spinal fluid. J. Infect. Dis. 140:453-464. 7. Mitruka, B. M., R. S. Kundargi, and A. M. Jonas. 1972. Gas chromatography for rapid differentiation of bacterial infections in man. Med. Res. Eng. 11:7-11.
fatigue with associated weakness, decreased appetite, and weight loss since June of 1980. The patient had been seen as an outpatient on two separate occasions in June and July; laboratory work had revealed a mild leukopenia with lymphocytosis, normal sedimentation rate, and three negative blood cultures. A workup for Brucella had not been requested on the blood cultures. Upon admission, blood cultures for
Brucella were requested twice on the first day and with any temperature elevation thereafter. Five blood cultures were drawn in a three-day period. Each time 5 ml of blood were added to a Difco 50 ml tryptic soy broth blood culture bottle; a permanent vent was inserted, and the bottles were incubated at 37°C in a candle jar. The bottles were subcultured every five days to a tryptic soy agar plate containing 50/o sheep blood. The
Case Report Brucellosis
Submitted by Lynn Richmond, M.S. St. John's Hospital St. Paul, Minnesota A 28-year-old white male was admitted to St. John's Hospital on August 19, 1980. A packing house employee in the hog-kill area for 10 years, he had been experiencing elevated temperature, chills, sweating,
4
W. L. Hewltl. 1978. Antimicrobial synergism in the therapy of gramnegative rod bacteremia. Chemotherapy 24:45-54. 2. Berenbaum, M. C. 1978. A method for testing for synergy with any number of agents. J. Infect. Dis. 137: 122-130. 3. Klastersky, J., R. Cappel, and D. Danean, 1972, Clinical significance of in vitro synergism between antibiotics in gram-negative infections. Antimicrob. Agents Chemother. 2:470-475. 4. KIastersky, J., C. Hensgens, and F, Meunier-Carpentier, 1976, Comparative effectiveness of combinations of amikacin with penicillin G and amikacin with carbenicillin in
5.
6. 7.
8.
gram-negative septicemia: Doubleblind clinical trial. J. Infect. Dis. 134(Suppl.):S433-S440. Klaslersky, J., F. Meunier-Carpentier, and J. Prevost. 1977. Significance of antimicrobial synergism for the outcome of gram-negative sepsis. Am. J. Med. Sci. 273:157-167. McCabe, W. R., and G. G. Jackson. 1965. Treatment of pyeIonephritis. N. Engl. J, Med. 272:1037-1044. Moellering, R. C., Jr., C. Wennersten, and A. N. Weinberg. 1971. Studies on antibiotic synergism against enterococci. I. Bacteriologic studies. J. Lab. Clin. Med, 77: 821-828. Norden, C. W., H. Wentzel, and E, Keleti. I979. Comparison of techniques for measurement of in vitro antibiotic synergism. J. Infect. Dis. 140:629-633.
9. Reyes, M. P., M. R. EI-Khatib, W..t. Brown, F. Smith, and A. M. Lerner. 1979. Synergy between
carbenicillin and an aminoglycoside (gentamicin or tobramycin) against Pseudomonas aeruginosa isolated from patients with endocarditis and sensitivity of isolates to normal human serum, J. Infect. Dis, 140: 192-202. 10. Young, L. S. 1978. Review of clinical significance of synergy in gramnegative infections at the University of California Los Angeles Hospital. Infection 6(Suppl. 1~:$47-$52
Editorial
Direct Detection of Infections Using Gas Liquid Chromatography
Timothy E. Kiehn, Ph.D. Diagnostic Microbiology Laboratory Memorial Hospital New York, New York 10021 The direct analysis by gas-liquid chromatograph]/(GLC) of tissue homogenates, pus, and body fluids offers the possibility for a more rapid diagnosis of infectious diseases. GLC techniques were first described in the 1940s, and significant advances in techniques and instrumentation have occurred since. Initially, gas chromatography procedures were used primarily by the analytical chemist. The range of compounds that can be analyzed was greatly broadened by the development of procedures for the formation of volatile derivatives. GLC allows separation, quantitation, and tentative identification of many compounds of biological interest. In the 1960s microbiologists first utilized this capability in the identification of fermentation products and as an aid in bacterial classification. Multicomponent analysis of bacterial byproducts is now a routine procedure used as an aid in anaerobe identification. These techniques, however, ii
require that the organism first be isolated and cultured before the cells or the media are analyzed by GLC. The more desirable procedure of directly analyzing patient fluids or tissues for detection and identification of infectious agents was first studied in the late 1960s. Progress in this area has been less rapid due to the complexity of clinical material. In the ensuing 10 years, further advances in instrumentation and techniques, and a series of subsequent diagnostic successes have been made. A variety of column materials, derivatives, and detection methods are now available. Reliable and inexpensive instruments with thermal conductivity detectors are commonly used in the anaerobic laboratory and for the determination of lactic acid in cerebrospinal fluid (CSF). More sensitive flame-ionization and electron-capture detectors are usually necessary for more complex examinations. Fluids may be examined for the presence of single compounds, or an analysis of multiple compounds or "peaks" may be conducted. When examining complex chromatograms (fingerprint analysis) it is helpful to use an integrator for the evaluation of retention times and peak areas. Also, the GLC may be linked to a computer for collation and interpretation of the
chromatograms. Automatic samplers are also available, which allow analysis to continue after working hours. More specific identification of the compounds isolated can be achieved with the use o f a mass spectrometer. Direct tissue or body fluid analysis has been directed primarily at the diagnosis of respiratory infections, meningitis, arthritis, and septicemia. Knowledge gained in anaerobe identification has been utilized in the direct analysis of serous fluid or pus for the detection of fatty acids produced by anaerobic bacteria (4). These acids, which are products of bacterial metabolism, are present in increased quantities distinct from normal or background levels. For example, increased amounts of succinic acid have been found in clinical material from infections by Bacteroides fragilis. The early detection of this penicillin-resistant anaerobe is of clinical importance. GLC has been used to detect increased levels of lactic acid in CSF (1). Levels of this acid are increased during bacterial but not viral meningitis. The technique is also useful in monitoring therapy because the lactic acid level returns to normal as the organism is cleared. Tuberculous, cryptococcal, viral (3), and bacterial
(6) meningitis have also been differentiated by comparison of the CSF chromatographic pattern, sometimes with computer assistance. Bacteria commonly causing septic arthritis, such as staphylococci, streptococci, and neisseriae, have been directly identified in synovial fluids by electron-capture GLC (2). There have been few reports of the direct diagnosis of septicemia by GLC (7). This is due to the complexity of the matrix, the sensitivi.ty of the analysis required, and the difficulty of assembling well-characterized patient groups. Complex pattern comparison has met with little success; a more realistic approach may be metabolite detection, such as the increased arabinitol levels we have reported in invasive candidiasis (5). What are the advantages and difficulties associated with the use of GLC in the diagnostic microbiology laboratory? There are several important advantages to this procedure. It is rapid; most analyses of body fluids or tissues can be performed within one to two hours. Cultures take at least one day, and gram stains, although rapid, are not always helpful. GLC results are usually not affected by recent prior antimicrobial therapy, while culture growth may be inhibited by the antibiotic. GLC may be used to monitor therapy because microbial metabolites may disappear or return to normal levels with appropriate therapy. Other rapid diagnostic procedures have limitations. Immunologic techniques that
rely on detection of antibody may not provide early diagnosis or may not be helpful in immunocompromised patients. Countercurrent immunoelectrophoresis requires high-titered specific antisera, and reports indicate a 10-20% false-negative rate. The limulus assay for the diagnosis of bacterial meningitis is restricted to gram-negative organisms. Disadvantages or problems with GLC must also be considered. Initial cost of the equipment is certainly a factor. In some cases, as with lactic acid levels in meningitis, available enzyme assays are as reliable as GLC and do not require the use of expensive equipment. Training of personnel requires considerable time. The infecting organism must produce compounds that are detectable by the procedures employed. For example, not all strains of Bacteroides produce succinie acid. Much of the research thus far has been performed by only a few groups of investigators. Other laboratories must contribute information so that the reliability of these procedures may be determined. The use of GLC for the direct detection of infections is a promising field. However, significant advances in the use of GLC and other analytic techniques such as high-pressure liquid chromatography will only be made when we increase our collaboration with our colleagues in other technical disciplines. For example, our laboratory's success with GLC in the detection of Candida metabolites didn't begin until we started talking to competent biochemists.
References 1. Brook, I., K. S. Bricknell, G. D. Overturf, and S. M. Finegold. 1978. Measurement of lactic acid in cerebrospinal fluid of patients with infections of the central nervous system. J. Infect. Dis. 137:384-390. 2. Brooks, J. B., D. S. Kellog, C. C. Alley, H. B. Short, H. H. Handsfield, and B. Huff. 1974. Gas chromatography as a potential means of diagnosing arthritis. I. Differentiation between staphylococcal, streptococcal, gonococcal, and traumatic arthritis. J. Infect. Dis. 129:660-668. 3. Craven, R. B., J. B. Brooks, D. C. Edman, J. D. Converse, J. Greenlee, D. Schlossberg, T. Furlow, J. M. Gwaltney, Jr., and W. F. Miner. 1977. Rapid diagnosis of lymphocytic meningitis by frequency-pulsed electron capture gas-liquid chromatography: Differentiation of tuberculous, cryptococcal, and viral meningitis. J. Clin. Microbiol. 6:27-32. 4. Gorbach, S. L., J. W. Mayhew, J. G. Bartlett, H. Thadepalli, and A. B. Onderdonk. 1976. Rapid diagnosis of anaerobic infections by direct gasliquid chromatography of clinical specimens. J. Clin. Invest. 57:478-484. 5. Kiehn, T. E., E. M. Bernard, J. W. M. Gold, and D. Armstrong. 1979. Candidiasis: Detection by gas-liquid chromatography of D-arabinitol, a fungal metabolite, in human serum. Science 206:577-580. 6. La Force, F. M., J. L. Brice, and T. G. Tornabene. 1979. Diagnosis of bacterial meningitis by gas-liquid chromatography. II. Analysis of spinal fluid. J. Infect. Dis. 140:453-464. 7. Mitruka, B. M., R. S. Kundargi, and A. M. Jonas. 1972. Gas chromatography for rapid differentiation of bacterial infections in man. Med. Res. Eng. 11:7-11.
fatigue with associated weakness, decreased appetite, and weight loss since June of 1980. The patient had been seen as an outpatient on two separate occasions in June and July; laboratory work had revealed a mild leukopenia with lymphocytosis, normal sedimentation rate, and three negative blood cultures. A workup for Brucella had not been requested on the blood cultures. Upon admission, blood cultures for
Brucella were requested twice on the first day and with any temperature elevation thereafter. Five blood cultures were drawn in a three-day period. Each time 5 ml of blood were added to a Difco 50 ml tryptic soy broth blood culture bottle; a permanent vent was inserted, and the bottles were incubated at 37°C in a candle jar. The bottles were subcultured every five days to a tryptic soy agar plate containing 50/o sheep blood. The
Case Report Brucellosis
Submitted by Lynn Richmond, M.S. St. John's Hospital St. Paul, Minnesota A 28-year-old white male was admitted to St. John's Hospital on August 19, 1980. A packing house employee in the hog-kill area for 10 years, he had been experiencing elevated temperature, chills, sweating,
4