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.     

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.     

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.     

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.     

Diagnostic histopathologique de la mononucléose infectieuse

Infectious mononucleosis corresponds to the clinical syndrome of primary infection by Epstein-Barr virus (EBV). When diagnosis has not been achieved, pathologists may have to analyze lymph node biopsy, tonsil sample or spleen. In all cases, the diagnosis is based on a massive proliferation of infected B cells and cytotoxic T cells. Tissue architecture is preserved, sometimes masked by the polymorphic cellular infiltration, comprising many cells with plasmacytic differentiation. Immunohistochemistry shows the mixture of B and T immunoblasts, the polytypic phenotype of cells with plasmacytic differentiation and numerous cytotoxic T cells. EBV is evidenced by in situ hybridization (EBER-1 probe) or by immunohistochemistry with detection of latent viral proteins (LMP-1, EBNA-2). Differential diagnosis can be difficult with some lymphomas (Hodgkin's disease, Diffuse large B cell lymphomas…) as well as with other infectious chronic lymphadenopathies.