PCR-RFLP as a useful tool for diagnosis of invasive mycoses in a healthcare facility in the North of Brazil

Background The incidence of invasive mycoses is increasing worldwide. PCR-RFLP was applied to the identification of 10 reference strains and 90 cultures of agents of invasive mycoses. In addition, the new approach was applied to detect fungal agents in 120 biological samples (blood, cerebrospinal fluid and bone marrow). PCR-RFLP results were compared with the ones obtained with conventional methods (culture, microscopy, and biochemical testing). Results The assays carried out with the reference strains (Candida albicans, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Cryptococcus neoformans, Cryptococcus gattii and Histoplasma capsulatum), demonstrated that the RFLP profiles were correctly predicted by the in silico investigation and allowed unequivocal identification of all chosen reference strains. The PCR-RFLP also identified 90 cultures of agents of invasive mycoses correctly, 2.5 times faster than the conventional assays. Evaluating PCR-RFLP with biological samples it was observed that the PCR was found to be 100% accurate and the RFLP profiles allowed the identification of the etiological agents: C. neoformans (n = 3) and C. gattii (n = 1) in CSF samples, H. capsulatum (n = 1) in bone marrow and C. albicans (n = 2) in blood cultures. The detection and identification by PCR-RFLP were found to be between two to ten times faster than the conventional assays. Conclusion The results showed that PCR-RFLP is a valuable tool for the identification of invasive mycoses that can be implemented in hospital laboratories, allowing for a high number of clinical analyses per day.

Antimicrobial properties of magnesium chloride at low pH in the presence of anionic bases.

Magnesium is an element essential for life and is found ubiquitously in all organisms. The different cations play important roles as enzymatic co-factors, as signaling molecules, and in stabilizing cellular components. It is not surprising that magnesium salts in microbiological experiments are typically associated with positive effects. In this study with Listeria monocytogenes as a model organism, we focus however on the usefulness of magnesium (in form of MgCl2) as a stress enhancer. Whereas MgCl2 does not affect bacterial viability at near-neutral pHs, it was found to strongly compromise culturability and redox activity when cell suspensions were exposed to the salt at acidic pH. The principle was confirmed with a number of gram-negative and gram-positive species. The magnesium salt dramatically increased the acidity to a level that was antimicrobial in the presence of anionic bases such as phosphate, lactate, or acetate, but not TRIS. The antimicrobial activity of MgCl2 was much stronger than that of NaCl, KCl, or CaCl2. No effect was observed with MgSO4 or when cells were exposed to MgCl2 in phosphate buffer with a pH ≥ 5. Acid stress was reinforced by an additional, salt-specific effect of MgCl2 on microbial viability that needs further examination. Apart from its implications for surface disinfection, this observation might support the commonly stated therapeutic properties of MgCl2 for the treatment of skin diseases (with healthy skin being an acidic environment), and could contribute to understanding why salt from the Dead Sea, where Mg(2+) and Cl(-) are the most abundant cation/anion, has healing properties in a microbiological context.

Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment.

Treatment of microbiological samples with viability dyes prior to extraction of DNA and PCR amplification for downstream analysis has evolved into a commonly applied method. The addition of this easy-to-perform step to the sample analysis procedure inhibits the amplification of DNA from dead cells with compromised cell membranes. The method is currently used both in combination with quantitative PCR (qPCR), end-point PCR, and isothermal amplification. We present here a detailed study of the effect of amplicon size on amplification signals from unstressed and heat-exposed cells after treatment with propidium monoazide (PMA). PMA treatment was shown to be more efficient in excluding dead cells from the analysis both in combination with qPCR (PMA-qPCR) and denaturing gradient gel electrophoresis (PMA-DGGE), when longer amplicons were used. When applied to pure cultures of the fish pathogens Vibrio anguillarum and Flavobacterium psychrophilum exposed to a heat gradient ranging from mild to lethal, qPCR product lengths did not influence PMA-qPCR results at low temperatures, whereas an increasingly strong impact was seen at higher temperatures. Membrane permeability as a result of heat exposure might however have to be considered a conservative parameter for cell death for these pathogens as culturability and redox activity were lost at lower stress intensities than membrane integrity. When applying PMA-DGGE to an environmental water sample which was either left untreated or was exposed to heat, differences to non-PMA treated samples tended to slightly increase when amplified fragments in the first round of the nested PCR were longer, whereas the impact of 1st-round cycle numbers remains unclear.