NDBD

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NDBD

  1. 1. Indian Journal of Pediatrics, Volume 74—July, 2007 673 Correspondence and Reprint requests : Prof. B.D. Bhatia, Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221005, India [Received June 20, 2007; Accepted June 20, 2007] Symposium : Newer Diagnostic Tests Evaluation of patients with the signs and symptoms of bacterial sepsis requires a combination of clinical acumen and laboratory support. The diagnosis of bacterial infection can be particularly difficult in certain patients, such as neonates and young infants. Blood culture is widely used for the diagnosis of septicemia.However, in many clinical situations the yield from blood culture is low, positive cultures are obtained from fewer than 30%. Some of the patients with false-negative blood culture may have had prior antibiotic treatment or they were not bacteremic at the time of blood collection. Consequently, considerable effort has been devoted to the development of rapid, sensitive and specific assays for the detection of causative organisms. In the last few years many new techniques have entered into the field of early diagnostic process of bacterial infections which we have subdivided further into three broad headings as described below: I. Direct demonstration of bacteria by identifying bacterial genomes II. Demonstration of indirect evidences of bacterial infection by identifying serological markers of systemic inflammatory response syndrome III. Rapid bacterial culture methods I. DEMONSTRATION OF BACTERIA BY IDENTIFYING BACTERIAL GENOMES During the past decade, there has been unprecedented progress in molecular biology as well as in the application of nucleic acid technology to the study of the epidemiology of human infection, some of which are summarized below. 1. Polymerase Chain Reaction: Today, the most widely used nucleic acid amplification and detection method, the Polymerase Chain Reaction (PCR) assay, has been found to have a substantial impact on the diagnosis of infectious diseases.1,2 The ability of this method to amplify minute amounts (less than 3 copies) of specific microbial DNA sequences in a background mixture of host DNA makes it a powerful diagnostic tool.3 Nucleic acid amplification is performed in a thermocycler, which is an instrument that can hold the assay’s reagents and allows the reactions to occur at the various temperatures required. In the initial step of the procedure, nucleic acid (e.g., DNA)is extracted from the microorganism or clinical specimen of interest. Heat (90ºC-95ºC) is used to separate the extracted double- stranded DNA into single strands (denaturation). Cooling to 55ºC then allows primers specifically designed to flank the target nucleic acid sequence to adhere to the target Newer Diagnostic Tests for Bacterial Diseases B.D. Bhatia and Sriparna Basu Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India ABSTRACT In diagnosing bacterial infections, the rapid identification of bacteremia at an early stage of the disease is critical for a favorable outcome. Furthermore, it is important that exact information be obtained on the stage of the disease rapidly in order to choose and initiate the appropriate therapy. In recent years many new techniques have been added in the diagnostic tools. During the past decade, there has been unprecedented progress in molecular biology as well as in the application of nucleic acid technology to the study of the epidemiology of human infection. Highly sensitive molecular techniques are found to be capable of detecting minute amounts of specific microbial DNA sequences and their complex genetic variations. Moreover, altered levels of biomarkers such as procalcitonin, C-reactive protein, tumor necrosis factor alpha, and several interleukins are also found to be promising to define systemic inflammatory response syndrome as indirect evidences of bacterial infections. Lastly, many rapid culture methods are coming up to achieve faster bacterial diagnosis. In this review we will focus on these three newer methods for the early diagnosis of bacterial infections. These approaches will help to expedite the diagnosis of especially early infections and might be a further step towards the improvement of therapeutic methods. [Indian J Pediatr 2007; 74 (7) : 673-677] E-mail : baldev_bhatia@rediffmail.com Key words : Bacterial infections; Sepsis; Polymerase chain reaction; Proinflammatory mediators; Culture 77
  2. 2. B.D. Bhatia and S. Basu 674 Indian Journal of Pediatrics, Volume 74—July, 2007 DNA (annealing). Following this, the enzyme Taq polymerase and nucleotides are added to create new DNA fragments complementary to the target DNA (extension).This completes one cycle of PCR. This process of denaturation, annealing and extension is repeated numerous times in the thermocycler. At the end of each cycle each newly synthesized DNA sequence acts as a new target for the next cycle, so that after 30 cycles millions of copies of the original target DNA are created. The result is the accumulation of a specific PCR product with sequences located between the 2 flanking primers.4 A major advantage of PCR is that it is far more sensitive than direct hybridization and thus requires extremely small amounts of target DNA as a template. PCR-based methods can detect as few as 10 to 100 copies of bacterial genome in clinical samples. The sample does not have to be highly purified, and even the starting DNA may be partially degraded as long as the target sequence (~100– 1000 bp) is intact. The rapidity of PCR makes it possible to generate detectable amounts of amplified target nucleic acid within hours, compared with several days by probe hybridization procedures and often weeks for the identification of many fastidious organisms by culture. Conventionally, the sequence of the 16S rRNA gene has been used to detect and identify bacterial infection in clinical practice. The concept of DNA microarray hybridization is introduced recently, and the technique has been applied in clinical diagnosis and many fields of research, e.g., functional genomics and genetic analysis. Microarrays are usually coupled with PCR where they serve as a set of parallel dot-blots to enhance products detection and identification.5 PCR assays that are currently available commercially for use in diagnostic laboratories include tests for the detection of Chlamydia trachomatis, C. pneumoniae, Mycobacterium tuberculosis, Mycoplasma pneumoniae and Neisseria gonorrhoeae.6 Multiplex PCR-based assays have been developed and have the advantage of detecting multiple pathogens in a single PCRreaction. These have been used to detect common bacterial causes of respiratory tract infections, bacteremia and meningitis. Another promising application of PCR is in the diagnosis of infectious conditions in which there are often too few organisms present for detection by other means (For example, for Borrelia burgdorferi in Lyme disease, the range of symptoms and the limited diagnostic methods available frequently delay the diagnosis).7 Assays have also been developed that can detect genomic sequences associated with antimicrobial resistance. PCR-based methods for the detection of antimicrobial resistance have been applied to bacteria including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and multidrug-resistant M. tuberculosis. Despite the obvious advantages to these newer procedures, there may be potential limitations to DNA amplification technology in the diagnostic microbiology laboratory. The accuracy and reproducibility of PCR assays depend on the technical expertise and experience of the operator. The extreme sensitivity of PCR paradoxically leads to one of its major drawbacks—the occurrence of false-positive results. Very small amounts of the amplified target sequence, of which up to 10 copies can be present in a single PCR solution, can contaminate laboratory equipment or reagents. The PCR product can even spread as airborne droplets in areas of sample or reagent preparation. This contaminating DNA can then serve as a template for further amplification, resulting in false-positive results in subsequent samples. For this reason laboratories should have separate rooms for different steps of the PCR procedure and must follow stringent quality control measures to prevent contamination or carry-over. False-negative test results may occur because of the presence of substances in the specimen that inhibit nucleic acid extraction or amplification. Certain specimen types (e.g., blood) are more likely to contain such inhibitors. The assays may also lack sensitivity if there is a low inoculum of the microorganism present in the clinical specimen. This may be exacerbated if an inadequate sample or very small specimen volume (i.e., < 20 µL) is available for testing. Another source of error is the detection of nonviable organisms by PCR, since it amplifies only a portion of the microbe’s genome. In such instances, the detection of complementary DNA by reverse-transcription PCR of messenger RNA encoded by the pathogenic organism can serve as evidence of active infection. Interpretation of nucleic acid amplification test results is not always clear- cut. For example, assays may detect the residual DNA of a pathogenic microorganism even after successful treatment, and it is not clear whether this represents the presence of a small number of viable organisms or amplified DNA from nonviable organisms. Therefore, PCR tests should not be used to monitorthe effectiveness of a course of therapy. Finally, it must be acknowledged that performance of a PCR assay is generally more expensive than conventional diagnostic laboratory methods. Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment. The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. The advantage of nested PCR is that if the wrong PCR fragment was amplified, the probability is quite low that the region would be amplified a second time by the second set of primers. Thus, Nested PCR is a very specific PCR amplification. ~ 78
  3. 3. Newer Diagnostic Tests for Bacterial Diseases Indian Journal of Pediatrics, Volume 74—July, 2007 675 2. Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analysis: Nucleic acid hybridization in its simplest form can be used in the detection of microorganisms or specific resistance genes. It confirms the results of microbial cultures or even detects organisms in clinical samples. This may require the extraction of DNA or, in some cases, RNA from a clinical sample (body fluid, peripheral white cells, aspirate or scraping, or fresh tissue). A labeled synthetic nucleic acid probe often less than 30 bases long can detect the presence of target nucleic acids (oligonucleotide probe). The oligonucleotide probes directly identify microbial genetic sequences in contrast to conventional immunologic tests and Western blot techniques, which pick up microbial gene products or proteins. Molecular hybridization has greater sensitivity and specificity than these conventional techniques. 3. RNA Typing or Ribotyping: RNA typing or ribotyping is another chromosomal detection technique. There is conservation of the genes that encode subunits of ribosomal RNA between species. Ribosomal genes are present throughout the chromosomes of bacteria: the sequences of DNA between the ribosomal genes vary in length. The digestion of chromosomal DNA by endonuclease produces random fragment polymorphic patterns when probed with ribosomal RNA. For this method, E coli 16S ribosomal RNA serves as a probe for the endonuclease-digested chromosomal DNA. Because chromosomal nucleotide patterns usually vary from strain to strain but not within a strain, this technique can identify organisms and differentiate strains. The technique has application in the typing of Haemophilus influenza, Pseudomonas capacia, E coli, Salmonella typhi, and Providencia stuartii.8 However, ribotyping is not as useful for gram-positive organisms; for instance, in enterococci, it does a little more than differentiate to a species level. 4. PCR Ribotyping: PCR ribotyping is the analysis of banding patterns obtained by gel electrophoresis of PCR- amplified fragments of the 16S to 23S ribosomal RNA intergenic spacer regions. It has considerable advantages in terms of speed and technical ease, and is useful in detecting and typing C difficile strains.9 5. Pulsed-Field Gel Electrophoresis: This is a widely used technique for analyzing a large amount of chromosomal DNA found in large bacterial chromosomal fragments generated by endonuclease digestion.10 6. Clamped Homogeneous Field Electrophoresis: Investigators have developed this method to compare large chromosomal fragments generated by restriction endonuclease digestion. This technique, a form of pulsed- field gel electrophoresis, may be an easy way to compare isolates of a species. Clamped homogeneous field electrophoresis helps in the analysis of vancomycin- resistant enterococci. No reliable typing system previously has been able to identify strains of this organism beyond the species level. This technique can type other bacteria, mycobacteria, and yeast as well. 7. Multilocus Sequence Typing : In principle, this is the genome-based version of the conventional method of multilocus enzyme electrophoresis. It helps in the typing of various bacterial species by identifying DNA alleles from various organisms, including Campylobacter jejuni, S aureus, and more recently, Enterococcus faecium. The method involves PCR amplification and the nucleic acid sequencing of multiple internal fragments of housekeeping genes. The advantages of this approach are that the culturing of pathogenic microorganisms is avoided, as their gene fragments are amplified directly from biologic samples, and that the sequencing data are unambiguous, easy to standardize, and electronically portable. 8. Fluorescence-Based Amplified Fragment Length Polymorphism: This is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fluorescence-based amplified fragment length polymorphism assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. It has been useful in the study of vancomycin-resistant enterococci.11 9. DNA Sequencing and Molecular Evolutionary Analysis: The sequencing of DNA refers to the enumeration of individual nucleotide base pairs along a linear segment of DNA. Single-stranded or double- stranded DNA generated by PCR can be used directly for DNA sequencing. Automation has made it possible to double the lengths of readable DNA sequences obtained after a single sequencing run to more than 400 to 800 bp. Recent studies of microbial populations have demonstrated substantial genetic diversity within microbial species on a very minute level. Organisms have relatively small generation times. They have evolutionary divergence, as with any other species. This is due to the accumulation of random, nonlethal mutations. These may include minute changes, including single base pair substitutions, the deletion of individual genes, or even the acquisition of DNA from other microbial species. Over time, these lead to substantial genetic diversity within microbial species and indicate that isolates of organisms have many genetically diverging lineages. Highly sensitive molecular techniques are capable of detecting single base pair substitutions and resolving the mechanism of underlying complex variation and epidemiology. II. INDIRECT EVIDENCES OF BACTERIAL INFECTION BY IDENTIFYING SEROLOGICAL MARKERS In 1991, the American College of Chest Physicians and the Society of Critical Care Medicine convened a Consensus 79
  4. 4. B.D. Bhatia and S. Basu 676 Indian Journal of Pediatrics, Volume 74—July, 2007 Conference in an attempt to provide a conceptual and practical framework todefine the systemic inflammatory response to infection, which is a progressive injurious process that falls under the generalized term sepsis. Sepsis is considered when there is a systemic response to a possible infection, termed as systemic inflammatory response syndrome (SIRS). The quest for surrogate biomarkers to define SIRS has identified several potential candidates. Markers such as procalcitonin, C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and several interleukins appeared promising in initial studies. Several of them are described below. 1. Cytokines : As soon as a bacterium enters the body, it is confronted with two lines of defense: a humoral line and a cellular line. The humoral factors comprise complement, antibodies, and acute-phase proteins. In the cellular line of defense, in particular the mononuclear cells (monocytes and macrophages) and the neutrophils are of great significance since these cells may recognize bacterial cell wall constituents directly or indirectly after complement and antibody bind to the bacterium and its constituents. Bacterial cell wall constituents such as lipopolysaccharide (LPS), peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of sepsis. These constituentsinteract in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituentswith receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators bythe bacterial constituents. Recognition of LPS or other bacterial componentsinitiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators like TNF-α, IL-1, IL-2, IL-6, IL-8, IL-10, IL-12, IL 18, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)- CSF, platelet-activating factor (PAF), chemokines, eicosanoids and anaphylatoxins C3a and C5a. Measurement of these cytokines can be taken as fingerprints of sepsis. The host inflammatory responses to gram-negative and gram-positive stimuli share some common response elements but also exhibit distinct patterns of cytokine appearance and leukocyte gene expression. There are marked differences in the responses to gram-positive and gram-negative bacteria. Whereas, gram-negative bacteria all contain LPS as their major pathogenic determinant, gram-positive bacteria contain a number of immunogenic cell wall components besides the highly deleterious exotoxins. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10 and IL-18 binding protein in plasma do not differ between patients with sepsis due to gram- negative and gram-positive bacteria. However, plasma IL-1β, IL-6, and IL-18 concentrations are significantly higher in patients with sepsis due to gram-positive bacteria. The immunological response to gram-negative bacteria mainly involves leukocytesand the production of cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6. The release of exotoxins, many of which are superantigens, by gram-positive bacteria activates T cells, resulting in a different cellular response and different cytokine profile, with relatively lowlevels of TNF-α, IL-1, and IL-6 and increased levels of IL-8.9 Several studies have shown elevated plasma IL-18 concentrations to be associated with poor clinical out- come in severe inflammatory and septic conditions.12, 13 2. Procalcitonin (PCT): PCT is a relatively new marker that has been associated with inflammation and sepsis. It is a 116-amino-acid protein that is the precursor to calcitonin. The PCT plasma level in healthy individuals is low; usually below 0.1 ng/ml. The levels have been shown to rise with severity of sepsis. The sensitivity of PCT in initial determinations for the diagnosis of sepsis has been reported to vary from 61% to 85%, increasing to 72–100% within the subsequent 24 h. PCT specificity was found to vary in initial determinations from 50% to 97% and ranged between 63% and 97% within the next 24 hr.14 PCT had the highest sensitivity and specificity for differentiating SIRS from sepsis. It has been found to be a more reliable marker in the diagnosis of sepsis than other measures. 3. C-reactive protein (CRP): The acute phase protein CRP is released from the liver in response to proinflammatory cytokines and thought to recruit monocytes in early infection. Sensitivity and specificity of CRP levels in older children has been poor; however, in newborns CRP specificity averages 90%. Maximum CRP levels >3 mg/ dL had positive predictive values >20% for proven or probable early-onset infections and for proven or probable and proven late-onset infections, but only those >6 mg/dL had such a high positive predictive value for proven early-onset sepsis. Serial CRP levels are useful in the diagnostic evaluation of neonates with suspected infection. Two CRP levels <1 mg/dL obtained24 hr apart, 8 to 48 hours after presentation, indicate that bacterial infection is unlikely. The sensitivity of a normal CRP at the initial evaluation is not sufficient to justify withholding antibiotic therapy. The positive predictive value of elevatedCRP levels is low, especially for culture- proven early-onset infections.15 4. Cell surface receptors (neutrophil CD64 and CD11): Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis 80
  5. 5. Newer Diagnostic Tests for Bacterial Diseases Indian Journal of Pediatrics, Volume 74—July, 2007 677 compared with the standard diagnostic tests used in current medical practice with a sensitivity of 87.9% and specificity of 71.2%. Neutrophil CD64 is one of many activation-related antigenic changes manifested by PMNs during the normal pathophysiologic process of acute inflammatory response. Neutrophil expression of CD64 is upregulated under the influence of inflammation-related cytokines such as interleukin 12, interferon gamma, and granulocyte colony-stimulating factor. CD64 appears ideal as a surrogate marker of PMN activation or of systemic acute inflammatory response because its expression begins at less than 2000 sites per cell and becomes up-regulated in a graded fashion depending on the intensity of cytokines stimulation. Furthermore, the up-regulation of PMN CD64 expression appears to be of pathophysiologic significance because this form of Fc receptor has been shown to be functional in eliciting all the PMN functional responses used in antibacterial responses.16 Another cell surface receptor CD11 is also found to be upregulated during infection. III. RAPID CULTURE METHODS 1. BacT/Alert, BACTEC 660/730 and VITEK 2 nonradiometric blood culture systems: The detection of bloodstream infections is one of the most important tasks performed by the clinical microbiology laboratory. Rapid bacterial identification and susceptibility testing not only improve patient therapy and outcome, but also reduce costs. Both automated blood culture systems and automated systems for identification and susceptibility testing of bacteria have been on the market for a number of yr. These systems use fluorescence-based technology. They are used widely due to the characteristics such as lower contamination risks, shorter incubation periods and higher isolation rates. Though both rapid and sen- sitive, false-positive and false-negative results are high.17 2. The rapid evaluation of bacterial growth and antibiotic susceptibility in blood cultures by selected ion flow tube mass spectrometry: To achieve faster bacterial diagnosis, selected ion flow tube mass spectrometry (SIFT-MS) measured metabolic gases in the headspaces of BacT/ALERT blood culture bottles. Pseudomonas aeruginosa, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus and Neisseria meningitidis growth and trace gas patterns were detected from 10 colony forming units after 6 hours.18 In a recent study of ours (unpublished data) blood culture was positive only in 24 out of 55 cases of neonatal sepsis, whereas nested PCR was positive in 83.6% of suspected cases of neonatal sepsis. Even the primary PCR was positive only in 25 out of 55, whereas nested PCR was positive in 21 out of 30 cases where primary PCR was negative. In 15 suspects where all tests were negative, nested PCR was positive in 12 cases. REFERENCES 1. Naber SP. Molecular pathology: diagnosis of infectious disease. N Engl J Med 1994; 331 : 1212-1215. 2. Fredricks DN, Relman DA. Application of polymerase chain reactions to the diagnosis of infectious diseases. Clin Infect Dis 1999; 29 : 475-488. 3. Shang S, Chen Z, Yu X. Detection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemia. Acta Paediatr 2001; 90 : 179-183. 4. Louie M, Louie L, Simor AE. The role of DNA amplification technology in the diagnosis of infectious diseases. CMAJ (Can Med Assoc J 2000; 163 : 301-309. 5. Bischoff M, Dunmam P, Kormanec J et al. Microarray-based analysis of the Staphylococcus aureus sigma B regulon. J Bacteriol 2004; 186 : 4085-4099. 6. Tompkins LS. The use of molecular methods in infectious diseases. N Engl J Med 1992; 327 : 1290-1297. 7. Meloni R, Khalfallah O, Biguet NF. DNA microarrays and pharmacogenomics. Pharmacol Res 2004; 49 : 303-308. 8. Tenover FC. Molecular methods for the clinical microbiology laboratory. In: Balows A, ed. Manual of Clinical Microbiology. Washington, DC: American Society for Microbiology; 1991: 119-127. 9. Spigaglia P, Cardines R, Rossi S et al. Molecular typing and long-term comparison of Clostridium difficile strains by pulsed- field gel electrophoresis and PCR-ribotyping. J Med Microbiol 2001; 50 : 407-414. 10. Turabelidze D, Kotetishvili M, Kreger A et al. Improved pulsed-field gel electrophoresis for typing vancomycin- resistant enterococci. J Clin Microbiol 2000; 38 : 4242-4245. 11. Antonishyn NA, McDonald RR, Chan EL et al. Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium. J Clin Microbiol 2000; 38 : 4058-4065. 12. Tschoeke SK, Oberholzer A, Moldawer LL. Interleukin-18: a novel prognostic cytokine in bacteria-induced sepsis. Crit Care Med 2006 Apr; 34(4) : 1225-1233. 13. Feezor RJ, Oberholzer C, Baker HV et al. Molecular Characterization of the Acute Inflammatory Response to Infections with Gram-Negative versus Gram-Positive Bacteria Infect Immun 2003 October; 71(10): 5803-5813. 14. Chiesa C, Pellegrini G, Panero A et al. C-Reactive Protein, Interleukin-6, and Procalcitonin in the Immediate Postnatal Period: Influence of Illness Severity, Risk Status, Antenatal and Perinatal Complications, and Infection. Clinical Chemistry 2003; 49 : 60-68. 15. Benitz WE, Han MY, Madan A, Ramachandra P. Serial Serum C - reactive protein Levels in the Diagnosis of Neonatal Infection. Pediatr 1998 Oct; 102 (4) : 41-51. 16. Davis BH, Olsen SH, Ahmad E, Bigelow NC. Neutrophil CD64 is an improved indicator of infection or sepsis in emergency department patients. Arch Pathol Lab Med 2006 May; 130(5) : 654-661. 17. Kobayashi I, Yamamoto M, Hasegawa M, Sato Y, Uchino U, Kaneko A. Effect of delay of blood cultures on positive detection by automated blood culture system. Kansenshogaku Zasshi 2004 Nov; 78(11) : 959-966. 18. Scotter JM, Allardyce RA, Langford VS, Hill A, Murdoch DR. The rapid evaluation of bacterial growth in blood cultures by selected ion flow tube-mass spectrometry (SIFT-MS) and comparison with the BacT/ALERT automated blood culture system. J Microbiol Methods 2006 Jun; 65(3) : 628-631. 81

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