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doi: 10.1111/j.1600-0463.2004.apm11211-1202.xpmid: 15638835
Molecular diagnostic techniques, such as PCR, have become useful tools for the rapid etiological diagnosis of lower respiratory tract infections. Nucleic acid amplification tests (NAATs) have been evaluated for detecting most respiratory pathogens, and commercial assays are available for some pathogens. However, standardized protocols are needed before these assays are introduced into routine diagnostic use. For pneumonia, NAATs offer advantages over conventional tests for the detection of Mycoplasma pneumoniae, Legionella spp. and Chlamydia pneumoniae. For pneumococcal pneumonia in adults, PCR adds little to existing diagnostic tests, and is unable to distinguish pneumococcal colonization from infection when testing respiratory samples. Although less sensitive than culture‐based methods, several commercial molecular diagnostic assays have been developed for tuberculosis and are useful rapid tests for selected patients. PCR can now be considered the rapid diagnostic test of choice for pertussis and some respiratory virus infections. Further work is required to better characterize the role of molecular diagnostic tests for diagnosing lower respiratory tract infections, and to develop standard assays that can be readily adopted by routine diagnostic laboratories.
SHAMPUTA, I. C.; RIGOUTS AND, L.; PORTAELS, F.
doi: 10.1111/j.1600-0463.2004.apm11211-1203.xpmid: 15638836
Mycobacteria comprise a diverse group of bacteria that are widespread in nature, some of which cause significant disease in humans. Members of the Mycobacterium tuberculosis complex (MTBC) are the most important human pathogens of the genus Mycobacterium. Traditional methods for detection and identification of mycobacteria include microscopy, culture and phenotypic tests. These methods either lack sensitivity, specificity, or are time consuming. Advances in the field of molecular biology have provided rapid diagnostic tools that have reduced the turnaround times for detecting MTBC and drug resistance in cultures and directly in clinical specimens from weeks to days. This review discusses the molecular genetic techniques for detecting and identifying MTBC as well as drug resistance of mycobacteria in clinical specimens.
TAHA, MUHAMED‐KHEIR; OLCÉN, PER
doi: 10.1111/j.1600-0463.2004.apm11211-1204.xpmid: 15688522
Acute bacterial infection of the central nervous system requires rapid and adequate management. Etiological diagnosis is hence crucial. Moreover, the epidemic threat of certain bacteria necessitates a reliable characterization of the involved bacterial strains to follow the spread of epidemic strains. Conventional identification and characterization of etiological agents are basically based on culture and identification of bacterial markers most frequently by serological assays. Molecular identification and characterization of bacteria have been employed. They provide more reliable analysis of bacterial isolates. Molecular methods for non‐culture diagnosis of bacterial infections have recently been developed. In many cases, the molecular assays have decreased the identification time of positive cultures and rescued detection of pathogens in culture‐negative clinical samples.
FREDLUND, HANS; FALK, LARS; JURSTRAND, MARGARETHA; UNEMO, MAGNUS
doi: 10.1111/j.1600-0463.2004.apm11211-1205.xpmid: 15638837
One of the mainstays in the prevention of Chlamydia trachomatis and Neisseria gonorrhoeae infections is the availability of laboratory diagnostics with high sensitivity and specificity. Assays for diagnosis of C. trachomatis include cell culture and nucleic acid amplification tests (NAATs). The major target sequences for C. trachomatis diagnosis by NAATs are located at the cryptic plasmid and the major target used for characterisation is the omp1 gene. The gold standard for diagnosis of N. gonorrhoeae is culture. However, numerous NAATs for identification of N. gonorrhoeae and a number of molecular genetic methods for characterisation of N. gonorrhoeae have been developed. Probably no routine laboratory can attain as high sensitivity by culturing C. trachomatis or N. gonorrhoeae as by using NAATs. For that reason NAATs can be recommended for diagnosing C. trachomatis, but not as the only diagnostic assay for N. gonorrhoeae, due to lack of antibiotic susceptibility testing and specificity problems, most pronounced for pharyngeal and rectal samples. Genotyping of C. trachomatis or N. gonorrhoeae provides additional information for contact tracing. It is recommended for N. gonorrhoeae, at least in low prevalence geographic areas, but cannot today be recommended for C. trachomatis. This is due to the low genetic variability and hence the limited benefits for partner notification. However, genotyping of C. trachomatis may play an important role under special circumstances.
FENOLLAR, FLORENCE; RAOULT, DIDIER
doi: 10.1111/j.1600-0463.2004.apm11211-1206.xpmid: 15638838
Technological innovations in the detection and identification of microorganisms using molecular techniques such as polymerase chain reaction (PCR) have ushered in a new era with respect to diagnostic microbiology. PCR using universal or specific primers followed by identification of amplified product, mainly by sequencing, has enabled the rapid identification of cultured or uncultured bacteria. Thus, PCR may allow quick diagnosis of infections caused by fastidious pathogens for which culture could be extremely difficult. However, several pitfalls, such as false positives, have been observed with PCR, underlining the necessity to interpret the results obtained with caution. At present, certain improvements in the molecular genetic methods may be helpful for the diagnosis of infectious diseases. Indeed, the recent development of bacterial genome sequencing has provided an important source of potential targets for PCR, allowing rational choice of primers for diagnosis and genotyping. In addition, the development of new techniques such as real‐time PCR offers several advantages in comparison to conventional PCR, including speed, simplicity, reproducibility, quantitative capability and low risk of contamination. Herein, we review the general principles of PCR‐based diagnosis and molecular genetic methods for the diagnosis of several hard‐to‐culture bacteria, such as Rickettsia spp., Ehrlichia spp., Coxiella burnetii, Bartonella spp., Tropheryma whipplei and Yersinia pestis.
HOORFAR, JEFFREY; WOLFFS, PETRA; RÅDSTRÖM, PETER
doi: 10.1111/j.1600-0463.2004.apm11211-1207.xpmid: 15688523
Increased use of powerful PCR technology for the routine detection of pathogens has focused attention on the need for international validation and preparation of official non‐commercial guidelines. Bacteria of epidemiological importance should be the prime focus, although a “validation infrastructure” once established could easily be adapted for PCR‐based detection of viruses and parasites. The aim of standardization should be the widespread adoption of diagnostic PCR for routine pathogen testing. European experience provides the impetus for realization of this vision through preparation of quantitative reference DNA material and reagents, production of stringent protocols and tools for thermal cycler performance testing, uncomplicated sample preparation techniques, and extensive ring trials for assessment of the efficacy of selected matrix/pathogen detection protocols.
SUNDSFJORD, ARNFINN; SIMONSEN, GUNNAR S.; HALDORSEN, BJØRG C.; HAAHEIM, HÅKON; HJELMEVOLL, STIG‐OVE; LITTAUER, PIA; DAHL, KRISTIN H.
doi: 10.1111/j.1600-0463.2004.apm11211-1208.xpmid: 15638839
Accurate and rapid diagnostic methods are needed to guide antimicrobial therapy and infection control interventions. Advances in real‐time PCR have provided a user‐friendly, rapid and reproducible testing platform catalysing an increased use of genetic assays as part of a wider strategy to minimize the development and spread of antimicrobial‐resistant bacteria. In this review we outline the principal features of genetic assays in the detection of antimicrobial resistance, their advantages and limitations, and discuss specific applications in the detection of methicillin‐resistant Staphylococcus aureus, glycopeptide‐resistant enterococci, aminoglycoside resistance in staphylococci and enterococci, broad‐spectrum resistance to β‐lactam antibiotics in gram‐negative bacteria, as well as genetic elements involved in the assembly and spread of antimicrobial resistance.
doi: 10.1111/j.1600-0463.2004.apm11211-1209.xpmid: 15638840
Antimicrobial susceptibility testing has traditionally been based on measurements of minimal inhibitory concentrations of antimicrobials. Molecular genetic studies on antimicrobial resistance have produced a great deal of genetic information which can be used for diagnosis of antimicrobial resistance determinants. Bacteria can acquire resistance to macrolides, lincosamides and streptogramin antibiotics by modification of the target site of the drugs, by active efflux of the drugs, and by inactivation of the drugs. The genetic backgrounds of these resistance mechanisms are well known and several molecular methods for detection of resistance determinants have been developed. Outbreaks of multidrug‐resistant tuberculosis have focused international attention on the emergence of Mycobacterium tuberculosis strains that are resistant to antimycobacterial agents. Knowledge of the antimycobacterial resistance genetics and progress in molecular methods has made it possible to develop rapid molecular methods for susceptibility testing. This review presents the genetic background of drug resistance and introduces some methods for genotypic susceptibility testing.
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