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Chapter 12.1 : Introduction

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Abstract:

Molecular techniques have been used successfully to aid in the detection and identification of pathogens and for the treatment of many infectious diseases ( ). Some of the earliest diagnostic applications included direct nucleic acid probes for identification of bacterial or fungal isolates and for direct detection of microorganisms in patient samples. This was followed by the introduction of nucleic acid amplification techniques (NAATs) for which utility in clinical testing was originally complicated by the complexity of the testing processes. These processes included many manual steps, such as nucleic acid extraction, amplification, and detection, and methods were fraught with the potential for false-positive results due to a combination of contamination risks plus the exquisite sensitivity of the testing methods. With the acquisition of expertise and dissemination of information regarding appropriate molecular laboratory procedures, including the production of CLSI guidelines ( ) and the recent inclusion of all molecular infectious disease testing into the CAP microbiology checklist ( ), along with the advent of newer technologies, such as automated extraction and real-time detection, use of molecular techniques in the clinical setting has become more commonplace and is no longer considered to be an esoteric or specialized practice reserved for reference laboratories. This is best exemplified by the fact that molecular testing systems are now available for use in moderate-complexity laboratory settings.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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References

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1. Bergeron, M. G.,, and M. Ouellette. 1998. Preventing antibiotic resistance using rapid DNA-based diagnostic tests. Infect. Control Hosp. Epidemiol. 19:560564.
2. Clarridge, J. E. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin. Microbiol. Rev. 17:840862.
3.College of American Pathologists. 2009. Laboratory Accreditation Program, Microbiology Checklist, rev. 06/15/2009. College of American Pathologists, Northfield, IL.
4. Cormican, M. G.,, and M. A. Pfaller,. 2001. Molecular pathology of infectious diseases, p. 12411253. In J. B. Henry (ed.), Clinical Diagnosis and Management by Laboratory Methods, 20th ed. W. B. Saunders Company, Philadelphia, PA.
5. Hacek, D. M.,, T. Suriano,, G. A. Noskin,, J. Kruszynski,, B. Reisberg,, and L. R. Peterson. 1999. Medical and economic benefit of a comprehensive infection control program that includes routine determination of microbial clonality. Am. J. Clin. Pathol. 111:647654.
6. Lakeman, F. D.,, and R. J. Whitley. 1995. Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease. J. Infect. Dis. 171:857863.
7. Nolte, F. S.,, and A. M. Caliendo,. 2007. Molecular detection and identification of microorganisms, p. 218244. In P. R. Murray,, E. J. Baron,, J. H. Jorgensen,, M. L. Landry,, and M. A. Pfaller (ed.), Manual of Clinical Microbiology, 9th ed. ASM Press, Washington, DC.
8. Nolte, F. S.,, F. R. Cockerill,, P. J. Dailey,, D. Hillyard,, S. McDonough,, R. F. Meyer,, and R. G. Shively. 2006. Clinical and Laboratory Standards Institute (CLSI). Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline—Second Edition. CLSI, Wayne, PA.
9. Nolte,, Persing, D. H. (ed.). 1996. PCR Protocols for Emerging Infectious Diseases. ASM Press, Washington, DC.
10. Persing, D. H.,, F. C. Tenover,, J. Versalovic,, Y. Tang,, E. R. Unger,, D. A. Relman,, and T. J. White (ed.). 2004. Molecular Microbiology: Diagnostic Principles and Practice. ASM Press, Washington, DC.
11. Pfaller, M. A. 1999. Molecular epidemiology in the care of patients. Arch. Pathol. Lab. Med. 123:10071010.
12. Pfaller, M. A. 2000. Diagnosis and management of infectious diseases: molecular methods for the new millennium. Clin. Lab. Newsl. 26:1013.

Tables

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Table 12.1-1

Commercially available, FDA-cleared nucleic acid probe hybridization methods for direct pathogen detection in clinical specimens

This table contains examples of FDA-cleared or -approved commercially available kits and is not intended to be all-inclusive. Abbreviations: HCV, hepatitis C virus; bDNA, branched-chain DNA.

San Diego, CA.

Sparks, MD.

Gaithersburg, MD.

Tarrytown, NY.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-2a

Commercially available, FDA-cleared nucleic acid amplification tests

This table contains examples of FDA-cleared or -approved nucleic acid amplification tests that are commercially available in the United States. It is not intended to be all-inclusive and does not contain tests used for screening of blood products. Websites of the manufacturers are useful sources of the most up-to-date information. Abbreviations: RT-PCR, reverse transcriptase PCR; SDA, strand displacement amplification; TMA, transcription-mediated amplification; NASBA, nucleic acid sequence-based amplification; TSPE, target-specific primer extension; MRSA, methicillin-resistant S. aureus; HBV and HCV, hepatitis B and C viruses, respectively; HIV-1, HIV type 1.

See Table 12.2.3-1 for additional details of collection devices for and .

Sparks, MD.

Branchburg, NJ; distributed by Roche Diagnostics Corp., Indianapolis, IN.

Des Plaines, IL.

San Diego, CA.

Sunnyvale, CA.

Durham, NC.

Tarrytown, NY.

Waukesha, WI.

Toronto, Canada.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
Generic image for table
Table 12.1-2b

Commercially available, FDA-cleared nucleic acid amplification tests

This table contains examples of FDA-cleared or -approved nucleic acid amplification tests that are commercially available in the United States. It is not intended to be all-inclusive and does not contain tests used for screening of blood products. Websites of the manufacturers are useful sources of the most up-to-date information. Abbreviations: RT-PCR, reverse transcriptase PCR; SDA, strand displacement amplification; TMA, transcription-mediated amplification; NASBA, nucleic acid sequence-based amplification; TSPE, target-specific primer extension; MRSA, methicillin-resistant S. aureus; HBV and HCV, hepatitis B and C viruses, respectively; HIV-1, HIV type 1.

See Table 12.2.3-1 for additional details of collection devices for and .

Sparks, MD.

Branchburg, NJ; distributed by Roche Diagnostics Corp., Indianapolis, IN.

Des Plaines, IL.

San Diego, CA.

Sunnyvale, CA.

Durham, NC.

Tarrytown, NY.

Waukesha, WI.

Toronto, Canada.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-2c

Commercially available, FDA-cleared nucleic acid amplification tests

This table contains examples of FDA-cleared or -approved nucleic acid amplification tests that are commercially available in the United States. It is not intended to be all-inclusive and does not contain tests used for screening of blood products. Websites of the manufacturers are useful sources of the most up-to-date information. Abbreviations: RT-PCR, reverse transcriptase PCR; SDA, strand displacement amplification; TMA, transcription-mediated amplification; NASBA, nucleic acid sequence-based amplification; TSPE, target-specific primer extension; MRSA, methicillin-resistant S. aureus; HBV and HCV, hepatitis B and C viruses, respectively; HIV-1, HIV type 1.

See Table 12.2.3-1 for additional details of collection devices for and .

Sparks, MD.

Branchburg, NJ; distributed by Roche Diagnostics Corp., Indianapolis, IN.

Des Plaines, IL.

San Diego, CA.

Sunnyvale, CA.

Durham, NC.

Tarrytown, NY.

Waukesha, WI.

Toronto, Canada.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-3

FDA-cleared nucleic acid probes for organism identification

This table contains examples of FDA-cleared or -approved nucleic acid probe tests that are commercially available in the United States. It is not intended to be all-inclusive. Websites of the manufacturers are useful sources of the most up-to-date information. Abbreviation: PNA FISH, peptide nucleic acid fluorescence in situ hybridization.

San Diego, CA.

Woburn, MA.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-4a

Clinically important pathogens tested for by noncommercial nucleic acid amplification-based tests

All tests use PCR. The list is not intended to be all-inclusive.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
Generic image for table
Table 12.1-4b

Clinically important pathogens tested for by noncommercial nucleic acid amplification-based tests

All tests use PCR. The list is not intended to be all-inclusive.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-5

Molecular methods for epidemiological typing of microorganisms

This table contains examples of available methods and applications and is not intended to be all-inclusive. It has been adapted from reference .

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-6

Application of molecular methods for detection of antimicrobial resistance

Adapted from reference . Abbreviations: bDNA, branched-chain DNA; RFLP, restriction fragment length polymorphism; SSCP, single-stranded conformational polymorphism; LIPA, line probe assay; TK, thymidine kinase; RT, reverse transcriptase; PROT, protease.

encodes the altered penicillin binding protein PBP2a; phenotypic methods may require 48 h of incubation or more to detect resistance and are less than 100% sensitive. Detection of has potential for clinical application in specific circumstances.

Vancomycin resistance in enterococci may be related to one of four distinct resistance genotypes of which and are of the most importance. Genotypic detection of resistance is useful in validation of phenotypic methods.

Penicillin and cephalosporin resistance in is due to the alteration of one or more penicillin binding proteins.

The genetic basis of resistance to β-lactam antimicrobial agents among is extremely complex. The and genes are the two most common sets of plasmid-borne β-lactamase genes. The presence of either a or gene implies ampicillin resistance. Variants of the and genes (extended-spectrum β-lactamases) may also encode resistance to a range of broad-spectrum cephalosporins and to monobactams.

is a very slow-growing organism. Four weeks or more may be required to obtain phenotypic susceptibility test results. Detection of resistance genes in has potential for clinical application in the short term.

There are no phenotypic methods sufficiently practical for routine clinical detection of resistance to antiviral agents. Genotypic methods represent a practical approach to the routine detection of antiviral resistance.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1
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Table 12.1-7

Suppliers of reagents used for laboratory-developed PCR tests

This table contains representative examples of vendors which supply reagents that can be used for the production of laboratory-developed PCR tests. It is not intended to be all-inclusive. In addition to the components listed above, analyte-specific reagents targeting specific organisms can be obtained from a variety of sources.

Citation: Garcia L. 2010. Introduction, p 266-274. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch12.1

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