Approche moléculaire de la résistance à la méticilline de Staphylococcus aureus

Staphylococcus aureus is one of the most common species met in medical microbiology laboratories. It causes many infectious diseases, and some of them are severe. Today, it can resist to many antibiotics, that were initially fully active, such as G or A group penicillin, by producing a penicillinase. Hospital strains can also resist to M group penicillins (methicillin, oxacillin) by modifying the β-lactam target: penicillin binding protein (PBP). Indeed, these strains produce a new PBP, called PBP 2a, of which affinity for β-lactams is much lower than the one of common Staphylococcus PBP. This PBP2a is encoded by mecA gene, of which expression is under control of several regulation genes. The expression of methicillin-resistance can be homogeneous or heterogeneous. Some strains can resist to methicillin using other mechanisms (penicillinase overproduction, methicillin-specific hydrolytic enzyme, PBP modifications). Particular methods are needed for the phenotypic detection of methicillin-resistance: antibiograms on NaCl-supplemented agar or incubated at 30 °C. Molecular detection of mecA gene by PCR permits to detect that resistance on strains for which phenotypic detection has failed.     

Diagnostic moléculaire de la résistance de Mycobacterium tuberculosis aux antituberculeux

The development of new technical tools for molecular biology has modified the methods used for the identification of resistance in Mycobacterium tuberculosis. Although the proportion method generally remains the reference method, more rapid and sophisticated techniques are now available, such as PCR-SSCP analysis, LiPA hybridization and DNA sequencing. At present, PCR-SSCP analysis is no longer used in routine. The INNO-LiPA assay, which is well adapted to routine, allows the rapid determination of rifampin resistance, but is not developed yet for detecting resistance to other antimycobacterial drugs. DNA sequencing is a powerful tool for the detection of resistance to rifampin, pyrazinamide and fluoroquinolones, but not to isoniazid. However, the technique requires specific facilities which are very costly. Finally, the development of high-density oligonucleotide arrays that can be used to rapidly examine large amounts of DNA sequences could allow to simultaneously investigate a large number of mechanisms of resistance. This new approach represents a very promising alternative for the rapid analysis of genomic regions involved in drug resistance.     

Apport de la biologie moléculaire au diagnostic de la tuberculose

Reducing delays in diagnosing tuberculosis should contribute to lower the lethality and to reduce the transmission. Microscopy examination is an easy to perform and rapid technique, but lacks sensitivity and specificity. Culture and biochemical identification are sensitive and specific, but require two to eight weeks DNA probes performed on solid or liquid media are very efficient for M. tuberculosis complex strains identification. Nucleic acid amplification techniques are available to directly detect the M. tuberculosis complex in clinical samples. Performances are variable. Very good results are obtained with experienced teams who scrupulously follow the specified guidelines.     

Apport de la biologie moléculaire au diagnostic bactériologique

The use of molecular biology in the clinical laboratories is limited by the efficiency and by the cost-effectiveness of the phenotypic methods. PCR and TMA are routine tests for the direct detection of Mycobacterium tuberculosis, Chlamydia trachomatis and Neisseria gonorrhoeae. Hybridization probes are used for the identification of bacterial cultures. DNA chips are promising techniques in middle term future. Universal PCR is used by an increasing number of laboratories for bacterial identification.     

Applications de la biologie moléculaire au diagnostic de la toxoplasmose et d'autres protozooses

In medical parasitology PCR-based methods are the most frequently molecular used tools. The best results have been obtained for diagnosis of congenital toxoplasmosis by parasite DNA detection on amniotic fluid. This technique is now currently employed but a recent european study showed the need of an external quality assurance sheme because of the lack of homogeneity in results obtained by this blinded control (aliquots made with amniotic fluid spiked with Toxoplasma). For others protozoa the interest and limits of the use of PCR as diagnosis tool are in question..     

Diagnostic moléculaire de Neisseria meningitidis

Culture-confirmed diagnosis of meningococcal infections is hindered by the failure to isolate Neisseria meningitidis following an early antibiotic treatment, which is highly recommended whenever meningococcal infection is suspected. Molecular methods for nonculture diagnosis of meningococcal infections have been recently developed and they usually use PCR-based detection of meningococcal DNA. Samples correspond most frequently to blood and cerebrospinal fluid (CSF), however, other samples can also be tested such as articular fluid and pericardiac fluid is observed. Several strategies are currently used but they usually a two-step process, the detection of meningococcal DNA and the prediction of serogroup (genogrouping). N. meningitidis is highly variable due to frequent horizontal DNA exchange between strains. Molecular approaches of typing allow a reliable tracking of meningococcal strains and a powerful epidemiological analysis.     

Apports et limites de la biologie moléculaire en mycologie médicale

For medical mycology, molecular biology is a fantastic tool for epidemiological investigation. Molecular typing of environmental and patient isolates allows to define the way of transmission and the origin of nosocomial fungal outbreaks (candidiasis, aspergillosis). For diagnosis, detection of fungal DNA by PCR is actually no superior compared to blood culture (Candida) or serum antigen detection (Aspergillus). For identification of species, the interest is quite limited because of good results obtained with classical methods (biochimical tests, microscopy).     

Mécanismes et méthodes de détection de la résistance de Staphylococcus aureus aux glycopeptides

For more than 30 years, glycopeptides remained active on Staphylococcus aureus strains. Nevertheless, the emergence of strains resistant to these antibiotics was feared. In the last five years, numerous and diverse glycopeptide-resistant strains were isolated. Two major mechanisms could explain this resistance. Gene transfer from vancomycine-resistant enterococci strains to S. aureus results in the acquisition of a high level resistance to glycopeptides in this species. For the strains with lower susceptibility to glycopeptides, the main mechanism seems to be related to an increased cell wall thickness, which may result in trapping of glycopeptide molecules far from their active sites. High level resistance, because of high minimal inhibitory concentrations, is easily detected in the clinical laboratory. On the other hand, the detection of S. aureus strains with lower susceptibility to glycopeptides is difficult. The E-Test® or the modified population analysis method are sensitive, but difficult to use in routine practice. The development of techniques and culture media allowing a systematic and performing detection is still controversial.     

Intérêt de la biologie moléculaire dans le diagnostic des infections vlrales du système nerveux central

The use of PCR and hybridization for the detection of viral nucleic acids in cerebrospinal fluid (CSF) currently represents the most reliable method for the meningoencephalitis diagnosis due to herpes viruses, enteroviruses, JC polyomavirus and HIV-1 primary infection. The molecular tools are less suitable for the postinfectious encephalitis associated with a systemic viral infection such as neurologic complications of measles, rubella, influenza and varicella, The speed and high sensitivity allow earlier diagnosis and specific treatment. The monitoring of the antiviral therapy is assessed by the development of quantitative amplification assays in CSF or by a negative PCR result. The pattern mutations conferring resistance and the genetic variability of viruses (HIV-1, Dengue virus) could be determined from different locations in the genome. Theses methods performed over the past several years as research tools have now a large scale application with the commercialization of some tests. But before becoming the first line diagnostic test, PCR of CSF could be conducted with control quality and evaluated protocols for avoiding false positive and negative results.