After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.Rifampin (rifampicin), isoniazid, and the fluoroquinolones are the most important initial drug markers for extensively drug-resistant Mycobacterium tuberculosis strains, defined as multidrug-resistant (MDR) isolates (resistant to both isoniazid and rifampin) with additional resistance to a fluoroquinolone and to one of the injectable drugs (2). The fluoroquinolones have become an essential part of treatment regimens for MDR tuberculosis (7,25). Due to their potency and safety, the newgeneration fluoroquinolones are now even being evaluated as first-line medications for tuberculosis (3,13,20). Wang et al. further suggested that routine fluoroquinolone resistance testing may have a clinical impact by showing a significant correlation between development of fluoroquinolone and first-line M. tuberculosis drug resistance in an area in which resistant strains are highly endemic (28).The spontaneous acquisition of DNA sequence mutations is the primary genetic basis for the development of M. tuberculosis drug resistance (14). Since sequences are highly conserved, certain mutations correlate well with phenotypic resistance, and a limited number of mutations account for the majority of phenotypic resistance to the important antituberculosis medications, various methods of genotypic testing have successfully been used for the rapid detection of M. tuberculosis resistance (16,22). The sites that most frequently contain mutations associated with phenotypic resistance, called resistance-determining regions (RDR), differ depending on the drug tested. Among rifampin-resistant isolates worldwide, 95 to 97% harbor mutations in the rifampin RDR, an 81-bp ...

The past decade has seen a surge in the development of a variety of molecular diagnostics designed to rapidly identify or characterize medically important microorganisms. We briefly review important advances in molecular microbiology, and then discuss specific assays that have been implemented in clinical microbiology laboratories throughout the country. We also discuss emerging methods and technologies that will soon be more widely used for the prompt and accurate detection of the agents of infectious diseases.

Cell blocks are a useful adjunct to smears and monolayer slides in the evaluation of cytologic specimens. Their preparation captures residual small tissue fragments, clots, and cellular sediment, providing additional morphologic information in diagnostically challenging cases. 1,2 In addition, the use of cell blocks enables further immunostaining of paraffin embedded sections. Like other methods of slide preparation, however, this procedure is potentially prone to contamination by organic and inorganic material such as pollen, insects, and fungal organisms. 3 We describe a pseudo-outbreak of budding yeast forms found on cell block slides related to extrinsic contamination of the cell block process. Index CaseA 29-year-old man developed a Staphylococcal lugdunensis infection following arthroscopic surgery of his knee. He was treated with an arthroscopic ''washout'' and intravenous antibiotics. Despite this, pain and swelling in his knee progressed and he underwent joint aspiration on June 30, 2008, for re-evaluation. The synovial fluid appeared grossly turbid and was sent ''stat'' for cytologic exam. The patient was immediately taken to the operating room for arthrotomy, synovectomy, and debridement. Inflamed and necrotic-appearing tissue from the site was sent for culture and histopathology. Cytology of the synovial fluid was reviewed ahead and showed marked acute inflammation. In addition, budding yeast forms were noted in the cell block which lacked pseudohyphae, measured 2-5 microns in diameter, and were present in both H&E and Gomori methenamine silver (GMS) stained sections obtained from the same cell block (Fig. 1). Intraoperative specimens likewise showed acute inflammation but no organisms were seen and cultures were negative. Because of this unexpected finding, the clinician asked to review the cell block with the pathologist. Gram positive cocci in clusters consistent with Staphylococcus sp. were seen in addition to the yeast (Fig. 2). The ThinPrep TM , processed from the synovial fluid separately, was negative for yeast forms. Furthermore, cell blocks from other patient specimens processed on the same day were noted to have the same fungal organisms. This prompted an epidemiologic investigation to identify the source of cell block contamination. MethodsThe cell block preparation process was investigated and interviews with various personnel from the Cytology Laboratory were conducted.Cell blocks are prepared in our laboratory according to the plasma-thrombin method. 1,4 Fresh body fluid arrives at the Cytology Laboratory in a sealed container. A portion of the sample is placed in a labeled 50 ml centrifuge tube for cell block preparation. The tube is centrifuged (5 min at 2,500 rpm) and the supernatant decanted from the cell button. A nonsterile pipette is used to obtain three to four drops of outdated human plasma which is then added to the cell button and mixed. The plasma is not sterilized and is refrigerated in a glass container between uses.Three to four drops of thrombin, using another nonsterile...

No abstract

No abstract