Point-of-Care-Monitoring – Mikrobiologische POC-Diagnostik

Zusammenfassung

Die Mortalität schwerer Infektionen konnte in den letzten Jahrzehnten nicht nachhaltig reduziert werden. Der schnellstmögliche Beginn einer adäquaten antibiotischen Therapie ist wichtig zur Senkung der Mortalität. Der Goldstandard der mikrobiologischen Diagnostik sind Pathogen kultivierende Verfahren, die nach 24 bis 48 Stunden Ergebnisse liefern. Daher werden Patienten mit lebensbedrohlichen systemischen Infektionen mit einer empirischen gewählten Antibiotikatherapie behandelt, die sich bei einem Teil der Patienten als unwirksam erweist. Aktuelle mikrobiologische Point-of-Care-Tests sind pathogenspezifisch. Damit eignen sie sich nicht für den Einsatz bei schweren systemischen Infektionen (Sepsis). Molekulare Nukleinsäurediagnostik, wie die Polymerase-Kettenreaktion (PCR), ermöglicht die Identifikation von Pathogenen und Resistenzen. Diese Methoden werden in der Routine genutzt, um die Auswertung positiver Kulturen zu beschleunigen. Darüber hinaus sind erste Systeme erhältlich, die PCR-basiert, eine Keimidentifizierung ermöglichen, sofern der Erreger in dem Test-Panel der 25 häufigsten Sepsis-Erreger enthalten ist. Diese Systeme haben einen Zeitbedarf von unter 6 Stunden und können einen wichtigen Beitrag zur schnelleren adäquaten Therapie leisten. Aktuell existiert keines dieser molekularen mikrobiologischen Verfahren als Point-of-Care-Tool, da die Methodik sehr komplex ist. Es ist aber anzunehmen, dass diese Methode weiter automatisiert wird, das detektierbare Keimspektrum zunimmt und zunehmend Resistenzgene identifiziert werden können. Damit ist von einer wachsenden Bedeutung für die mikrobiologische Diagnostik auszugehen.

Abstract

It is well known that the early initiation of a specific antiinfective therapy is crucial to reduce the mortality in severe infection. Procedures culturing pathogens are the diagnostic gold standard in such diseases. However, these methods yield results earliest between 24 to 48 hours. Therefore, severe infections such as sepsis need to be treated with an empirical antimicrobial therapy, which is ineffective in an unknown fraction of these patients. Today's microbiological point of care tests are pathogen specific and therefore not appropriate for an infection with a variety of possible pathogens. Molecular nucleic acid diagnostics such as polymerase chain reaction (PCR) allow the identification of pathogens and resistances. These methods are used routinely to speed up the analysis of positive blood cultures. The newest PCR based system allows the identification of the 25 most frequent sepsis pathogens by PCR in parallel without previous culture in less than 6 hours. Thereby, these systems might shorten the time of possibly insufficient antiinfective therapy. However, these extensive tools are not suitable as point of care diagnostics. Miniaturization and automating of the nucleic acid based method is pending, as well as an increase of detectable pathogens and resistance genes by these methods. It is assumed that molecular PCR techniques will have an increasing impact on microbiological diagnostics in the future.

Schlüsselwörter:

Point-of-Care - mikrobiologische Diagnostik - PCR - Pathogene - Resistenzgene

Keywords:

point of care - microbiological tests - PCR - pathogens - resistance genes

Kernaussagen

• Das PIRO-Konzept der Sepsis ist ein Staging-Konzept, das eine umfassende Beurteilung des Zustandes erlaubt. Es ist mit den aktuellen Definitionen von Sepsis, schwerer Sepsis und septischem Schock nicht vergleichbar.

• Pathogen-kultivierende Methoden sind der Goldstandard der mikrobiologischen Diagnostik und bieten Ergebnisse nach 24–48 h.

• „Need for Speed“ ist eine zentrale Anforderung an mikrobiologische Diagnostik.

• Verfügbare immunchromatografische Point-of-Care-Tests sind Pathogen-spezifisch und eignen sich damit nicht für den Einsatz bei schweren systemischen Infektionen mit unbekannten Erregern.

• Der Einsatz der Polymerase-Kettenreaktion (PCR) kann den Zeitbedarf kultivierender Methoden verkürzen und die Sensitivität erhöhen.

• Aktuell existiert keine diagnostische molekulare mikrobiologische Point-of-Care-Anwendung.

• Die Automatisierung und Miniaturisierung wird höchstwahrscheinlich molekulare mikrobiologische Point-of-Care-Technik ermöglichen.

Literatur

• 1 Armstrong GL, Conn LA, Pinner RW. Trends in infectious disease mortality in the United States during the 20th century.  JAMA. 1999;  281 61-66
• 2 American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis.  Crit Care Med. 1992;  20 864-874
• 3 Levy MM et al.. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference.  Crit Care Med. 2003;  31 1250-1256
• 4 Dellinger RP et al.. Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock.  Intensive Care Med. 2004;  30 536-555
• 5 Garnacho-Montero J et al.. Impact of adequate empirical antibiotic therapy on the outcome of patients admitted to the intensive care unit with sepsis.  Crit Care Med. 2003;  31 2742-2751
• 6 Ibrahim EH et al.. The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting.  Chest. 2000;  118 146-155
• 7 Kumar A et al.. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock.  Crit Care Med. 2006;  34 1589-1596
• 8 Kuti EL, Patel AA, Coleman CI. Impact of inappropriate antibiotic therapy on mortality in patients with ventilator-associated pneumonia and blood stream infection: a meta-analysis.  J Crit Care. 2008;  23 91-100
• 9 Morrell M, Fraser VJ, Kollef MH. Delaying the empiric treatment of candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for hospital mortality.  Antimicrob Agents Chemother. 2005;  49 3640-3645
• 10 Sparwasser T et al.. Bacterial DNA causes septic shock.  Nature. 1997;  386 336-337
• 11 Gerber MA, Shulman ST. Rapid diagnosis of pharyngitis caused by group A streptococci.  Clin Microbiol Rev. 2004;  17
• 12 Roson B et al.. Contribution of a urinary antigen assay (Binax NOW) to the early diagnosis of pneumococcal pneumonia.  Clin Infect Dis. 2004;  38 222-226
• 13 Gutierrez F et al.. Evaluation of the immunochromatographic Binax NOW assay for detection of Streptococcus pneumoniae urinary antigen in a prospective study of community-acquired pneumonia in Spain.  Clin Infect Dis. 2003;  36 286-292
• 14 Murdoch DR et al.. Evaluation of a rapid immunochromatographic test for detection of Streptococcus pneumoniae antigen in urine samples from adults with community-acquired pneumonia.  J Clin Microbiol. 2001;  39 3495-3498
• 15 Shimada T et al.. Systematic review and metaanalysis: urinary antigen tests for Legionellosis.  Chest. 2009;  136 1576-1585
• 16 Honest H, Sharma S, Khan KS. Rapid tests for group B Streptococcus colonization in laboring women: a systematic review.  Pediatrics. 2006;  117 1055-1066
• 17 Bergeron MG, Ke D. New DNA-based PCR approaches for rapid real-time detection and prevention of group B streptococcal infections in newborns and pregnant women.  Expert Rev Mol Med. 2001;  3 1-14
• 18 Wolk DM et al.. Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares.  J Clin Microbiol. 2009;  47 758-764
• 19 Alcala L et al.. Comparison of three commercial methods for rapid detection of Clostridium difficile toxins A and B from fecal specimens.  J Clin Microbiol. 2008;  46 3833-3835
• 20 Bartlett JG, Gerding DN. Clinical recognition and diagnosis of Clostridium difficile infection.  Clin Infect Dis. 2008;  46 (S 01)
• 21 Nadala EC et al.. Performance evaluation of a new rapid urine test for chlamydia in men: prospective cohort study.  BMJ. 2009;  339
• 22 Murray CK et al.. Update on rapid diagnostic testing for malaria.  Clin Microbiol Rev. 2008;  21 97-110
• 23 Marx A et al.. Meta-analysis: accuracy of rapid tests for malaria in travelers returning from endemic areas.  Ann Intern Med. 2005;  142 836-846
• 24 Schuurman T et al.. Comparison of microscopy, real-time PCR and a rapid immunoassay for the detection of Giardia lamblia in human stool specimens.  Clin Microbiol Infect. 2007;  13 1186-1191
• 25 Oster N et al.. Evaluation of the immunochromatographic CORIS Giardia-Strip test for rapid diagnosis of Giardia lamblia.  Eur J Clin Microbiol Infect Dis. 2006;  25 112-115
• 26 Henrickson KJ, Hall CB. Diagnostic assays for respiratory syncytial virus disease.  Pediatr Infect Dis. 2007;  26
• 27 Foo H et al.. Laboratory test performance in young adults during influenza outbreaks at World Youth Day 2008.  J Clin Virol. 2009;  46 384-386
• 28 Storch GA. Rapid diagnostic tests for influenza.  Curr Opin Pediatr. 2003;  15 77-84
• 29 Wilhelmi I et al.. New immunochromatographic method for rapid detection of rotaviruses in stool samples compared with standard enzyme immunoassay and latex agglutination techniques.  Eur J Clin Microbiol Infect Dis. 2001;  20 741-743
• 30 Weitzel T et al.. Field evaluation of a rota- and adenovirus immunochromatographic assay using stool samples from children with acute diarrhea in Ghana.  J Clin Microbiol. 2007;  45 2695-2697
• 31 Branson BM. State of the art for diagnosis of HIV infection.  Clin Infect Dis. 2007;  45 (S 04)
• 32 Ninove L et al.. Impact of diagnostic procedures on patient management and hospitalization cost during the 2000 and 2005 enterovirus epidemics in Marseilles, France.  Clin Microbiol Infect. 2010;  16 651-656
• 33 Kost CB et al.. Multicenter beta trial of the GeneXpert enterovirus assay.  J Clin Microbiol. 2007;  45 1081-1086
• 34 Clerc O, Greub G. Routine use of point-of-care tests: usefulness and application in clinical microbiology.  Clin Microbiol Infect. 2010;  16 1054-1061
• 35 Briedigkeit L et al.. Recommendations of the German Working Group on medical laboratory testing (AML) on the introduction and quality assurance of procedures for Point-of-Care Testing (POCT) in hospitals.  Clin Chem Lab Med. 1999;  37 919-925
• 36 Meier FA, Jones BA. Point-of-care testing error: sources and amplifiers, taxonomy, prevention strategies, and detection monitors.  Arch Pathol Lab Med. 2005;  129 1262-1267
• 37 Weinstein MP et al.. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults.  Clin Infect Dis. 1997;  24 584-602
• 38 Leibovici L et al.. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection.  J Intern Med. 1998;  244 379-386
• 39 Kollef MH. Inadequate antimicrobial treatment: an important determinant of outcome for hospitalized patients.  Clin Infect Dis. 2000;  31 (S 04)
• 40 Harbarth S et al.. Inappropriate initial antimicrobial therapy and its effect on survival in a clinical trial of immunomodulating therapy for severe sepsis.  Am J Med. 2003;  115 529-535
• 41 Valles J et al.. Community-acquired bloodstream infection in critically ill adult patients: impact of shock and inappropriate antibiotic therapy on survival.  Chest. 2003;  123 1615-1624
• 42 Tumbarello M et al.. Bloodstream infections caused by extended-spectrum-beta-lactamase- producing Escherichia coli: risk factors for inadequate initial antimicrobial therapy.  Antimicrob Agents Chemother. 2008;  52 3244-3252
• 43 Struelens MJ, de Mendonça R. The emerging power of molecular diagnostics: towards improved management of life-threatening infection.  Intensive Care Med. 2001;  27 1696-1698
• 44 Weinstein MP et al.. The clinical significance of positive blood cultures in the 1990s: a prospective comprehensive evaluation of the microbiology, epidemiology, and outcome of bacteremia and fungemia in adults.  Clin Infect Dis. 1997;  24 584-602
• 45 Bates DW et al.. Predicting bacteremia in hospitalized patients. A prospectively validated model.  Ann Intern Med. 1990;  113 495-500
• 46 McKenzie R, Reimer LG. Effect of antimicrobials on blood cultures in endocarditis.  Diagn Microbiol Infect Dis. 1987;  8 165-172
• 47 Glerant JC et al.. Utility of blood cultures in community-acquired pneumonia requiring hospitalization: influence of antibiotic treatment before admission.  Respir Med. 1999;  93 208-212
• 48 Serody JS et al.. Utility of obtaining blood cultures in febrile neutropenic patients undergoing bone marrow transplantation.  Bone Marrow Transplant. 2000;  26 533-538
• 49 Krieg AM et al.. CpG motifs in bacterial DNA trigger direct B-cell activation.  Nature. 1995;  374 546-549
• 50 Pisetsky DS. Immune activation by bacterial DNA: a new genetic code.  Immunity. 1996;  5 303-310
• 51 Yamamoto S et al.. Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN (correction of INF) and augment IFN-mediated (correction of INF) natural killer activity.  J Immunol. 1992;  148 4072-4076
• 52 Hemmi H et al.. A Toll-like receptor recognizes bacterial DNA.  Nature. 2000;  408 740-745
• 53 Malouin F et al.. DNA probe technology for rapid detection of Haemophilus influenzae in clinical specimens.  J Clin Microbiol. 1988;  26 2132-2138
• 54 Davis TE, Fuller DD. Direct identification of bacterial isolates in blood cultures by using a DNA probe.  J Clin Microbiol. 1991;  29 2193-2196
• 55 Marlowe EM et al.. Application of an rRNA probe matrix for rapid identification of bacteria and fungi from routine blood cultures.  J Clin Microbiol. 2003;  41 5127-5133
• 56 Maes N et al.. Evaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative Staphylococci and determine methicillin resistance from blood cultures.  J Clin Microbiol. 2002;  40 1514-1517
• 57 Song JH et al.. Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction.  J Clin Microbiol. 1993;  31 1439-1443
• 58 Iralu JV et al.. Diagnosis of Mycobacterium avium bacteremia by polymerase chain reaction.  J Clin Microbiol. 1993;  31 1811-1814
• 59 Wheeler J et al.. PCR can add to detection of pneumococcal disease in pneumonic patients receiving antibiotics at admission.  J Clin Microbiol. 2000;  38 3907
• 60 Lorente ML et al.. Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study.  Thorax. 2000;  55 133-137
• 61 Zhang Y et al.. Detection of Streptococcus pneumoniae in whole blood by PCR.  J Clin Microbiol. 1995;  33 596-601
• 62 Guiver M. Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.  FEMS Immunol Med Microbiol. 2000;  28 173-179
• 63 Schluger NW et al.. Amplification of DNA of Mycobacterium tuberculosis from peripheral blood of patients with pulmonary tuberculosis.  Lancet. 1994;  344 232-233
• 64 Folgueira L et al.. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.  J Clin Microbiol. 1996;  34 512-515
• 65 Heininger A et al.. PCR and blood culture for detection of Escherichia coli bacteremia in rats.  J Clin Microbiol. 1999;  37 2479-2482
• 66 Wellinghausen N et al.. Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes.  Diagn Microbiol Infect Dis. 2004;  48 229-241
• 67 Klaschik S et al.. Detection and differentiation of in vitro-spiked bacteria by real-time PCR and melting-curve analysis.  J Clin Microbiol. 2004;  42 512-517
• 68 Wisplinghoff H et al.. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.  Clin Infect Dis. 2004;  39 309-317
• 69 Corless CE et al.. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.  J Clin Microbiol. 2001;  39 1553-1558
• 70 Klaschik S et al.. Real-time PCR for detection and differentiation of gram-positive and gram-negative bacteria.  J Clin Microbiol. 2002;  40 4304-4307
• 71 Lehmann LE et al.. A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples.  Med Microbiol Immunol. 2008;  197 313-324
• 72 Obaro SK, Madhi SA. Bacterial pneumonia vaccines and childhood pneumonia: are we winning, refining, or redefining?.  Lancet Infect Dis. 2006;  6 150-161
• 73 Rouphael NG et al.. A real-time polymerase chain reaction for the detection of Streptococcus pneumoniae in blood using a mouse model: a potential new "gold standard".  Diagn Microbiol Infect Dis. 2008;  62 23-25
• 74 Tarrago D et al.. Identification of pneumococcal serotypes from culture-negative clinical specimens by novel real-time PCR.  Clin Microbiol Infect. 2008;  14 828-834
• 75 Harris KA et al.. Duplex real-time PCR assay for detection of Streptococcus pneumoniae in clinical samples and determination of penicillin susceptibility.  J Clin Microbiol. 2008;  46 2751-2758
• 76 CDC-Centers for Disease Control and Prevention . Recommendations for preventing spread of vancomycin resistance. Recommendations of the Hospital Infection Control Practices Advisory Committee (HICPAC).  MMWR. 1995;  44 1-12
• 77 Mayhall CG. Prevention and control of vancomycin resistance in gram-positive coccal microorganisms: fire prevention and fire fighting.  Infect Control Hosp Epidemiol. 1996;  17 353-355
• 78 Stamper PD et al.. Clinical validation of the molecular BD GeneOhm StaphSR assay for direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures.  J Clin Microbiol. 2007;  45 2191-2196
• 79 van Hal SJ et al.. MRSA detection: comparison of two molecular methods (BD GeneOhm((R)) PCR assay and Easy-Plex) with two selective MRSA agars (MRSA-ID and Oxoid MRSA) for nasal swabs.  Eur J Clin Microbiol Infect Dis. 2009;  28 47-53
• 80 Kreger BE et al.. Gram-negative bacteremia. III. Reassessment of etiology, epidemiology and ecology in 612 patients.  Am J Med. 1980;  68 332-343
• 81 Werner AS et al.. Studies on the bacteremia of bacterial endocarditis.  JAMA. 1967;  202 199-203
• 82 Bottger EC. Frequent contamination of Taq polymerase with DNA.  Clin Chem. 1990;  36 1258-1259
• 83 Rand KH, Houck H. Taq polymerase contains bacterial DNA of unknown origin.  Mol Cell Probes. 1990;  4 445-450
• 84 Schmidt TM, Pace B, Pace NR. Detection of DNA contamination in Taq polymerase.  Biotechniques. 1991;  11 176-177
• 85 Jinno Y, Yoshiura K, Niikawa N. Use of psoralen as extinguisher of contaminated DNA in PCR.  Nucleic Acids Res. 1990;  18 6739
• 86 Sarkar G, Sommer SS. Shedding light on PCR contamination.  Nature. 1990;  343 27
• 87 Meier A et al.. Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.  J Clin Microbiol. 1993;  31 646-652
• 88 Klaschik S et al.. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR.  Mol Biotechnol. 2002;  22 231-242
• 89 Klaschik S et al.. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR.  Mol Biotechnol. 2002;  22 231-242
• 90 Kane TD, Alexander JW, Johannigman JA. The detection of microbial DNA in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients.  Ann Surg. 1998;  227 1-9
• 91 Sleigh J, Cursons R, La PM. Detection of bacteraemia in critically ill patients using 16S rDNA polymerase chain reaction and DNA sequencing.  Intensive Care Med. 2001;  27 1269-1273
• 92 Ley BE et al.. Detection of bacteraemia in patients with fever and neutropenia using 16S rRNA gene amplification by polymerase chain reaction.  Eur J Clin Microbiol Infect Dis. 1998;  17 247-253
• 93 Cursons RT, Jeyerajah E, Sleigh JW. The use of polymerase chain reaction to detect septicemia in critically ill patients.  Crit Care Med. 1999;  27 937-940
• 94 Kiechle FL, Holland CA. Point-of-care testing and molecular diagnostics: miniaturization required.  Clin Lab Med. 2009;  29 555-560