Combination of ß-(1, 3)-D-glucan testing in serum and qPCR in nasopharyngeal aspirate for facilitated diagnosis of Pneumocystis jirovecii pneumonia.

BACKGROUND: Currently, the biological diagnosis of Pneumocystis jirovecii pneumonia (PjP infection) usually relies on microbiological investigations in bronchial-alveolar lavage fluid (BALF) by conventional staining methods and/or molecular biology. However, bronchial-alveolar lavage is sometimes complicated to manage, especially in weakened patients. Therefore, alternative clinical samples-easier to collect-are warranted in such specific contexts. OBJECTIVE: Over a four-year period, diagnostic performance of an original method based on combination of quantitative real-time polymerase chain reaction (qPCR) in nasopharyngeal aspirate (NPA) with measurement of ß-(1, 3)-D-glucan antigen (BDG) in serum was prospectively assessed in a single centre. PATIENTS/METHODS: Results were compared with those obtained in BALF through direct staining methods and qPCR. True positives were defined by an independent committee based on clinical, radiological and biological data. Overall, 48 individuals with a definitive diagnosis of PjP infection were included, and 48 controls were selected upon matching for age, sex and underlying disease(s). RESULTS: qPCR results were strongly correlated between BALF and NPA (P < .0001). Altogether, greater diagnostic performance was achieved when establishing the positive cut-off of BDG antigen at 143 pg/mL. In such conditions, sensitivity of the testing based on either positive BDG measurement or positive qPCR in NPA was then calculated at 93.75%, 95% CI [82.37%-98.40%], and specificity at 97.87%, 95% CI [87.66%-100.00%]. CONCLUSIONS: Further validation through multicentre studies is now required, especially for establishing clear cut-offs. However, one could already state that combination of qPCR in the NPA with BDG measurement in serum may be a valuable substitute for BALF examination.

Cell-wall dyes interfere with Cryptococcus neoformans melanin deposition.

Melanization is an intrinsic characteristic of many fungal species, but details of this process are poorly understood because melanins are notoriously difficult pigments to study. While studying the binding of cell-wall dyes, Eosin Y or Uvitex, to melanized and non-melanized Cryptococcus neoformans cells we noted that melanization leads to reduced fluorescence intensity, suggesting that melanin interfered with dye binding to the cell wall. The growth of C. neoformans in melanizing conditions with either of the cell-wall dyes resulted in an increase in supernatant-associated melanin, consistent with blockage of melanin attachment to the cell wall. This effect provided the opportunity to characterize melanin released into culture supernatants. Released melanin particles appeared mostly as networked structures having dimensions consistent with previously described extracellular vesicles. Hence, dye binding to the cell wall created conditions that resembled the 'leaky melanin' phenotype described for certain cell-wall mutants. In agreement with earlier studies on fungal melanins biosynthesis, our observations are supportive of a model whereby C. neoformans melanization proceeds by the attachment of melanin nanoparticles to the cell wall through chitin, chitosan, and various glucans.

Use of calcofluor-blue brightener for the diagnosis of Pneumocystis jirovecii pneumonia in bronchial-alveolar lavage fluids: A single-center prospective study.

The biological diagnosis of Pneumocystis jirovecii pneumonia (PjP) is based on the investigation of respiratory fluids by conventional staining methods and/or molecular biology. Diagnostic performance of an in-house technique based on calcofluor-blue brightener for the direct detection of P. jirovecii cysts was prospectively assessed in bronchial-alveolar lavage fluids (BALF) from patients with a suspected PjP infection over a three-year period in a single center: the diagnostic yield was compared to that of a commercial kit based on monoclonal immunofluorescence assay (IFA) on replicate smears. May-Grünwald Giemsa (MGG) staining and quantitative Polymerase Chain Reaction (qPCR) were also performed. The gold standard for each patient was the definitive diagnosis of PjP infection by an independent committee based on clinical, radiological, and biological data. Overall, 481 BALF were assessed: 42 were found to be positive for the detection of P. jirovecii by at least one laboratory technique, but only 35 were actually judged to be in agreement with the definitive diagnosis of PjP infection. The sensitivity of the calcofluor-blue brightener technique was 74.3% vs. 60.0%, 34.6%, and 82.9% for IFA, MGG, and qPCR, respectively; and its specificity was 99.6% vs. 99.3%, 100.0%, and 99.4% for IFA, MGG, and qPCR. No technique was shown to be statistically superior to calcofluor-blue brightener. Further validation of the test through multicenter studies is now required, but in light of its low cost and easy preparation, the use of calcofluor-blue brightener in BALF appears to be a valuable alternative method for the routine first-line diagnosis of PjP infection.

Rapid protocol for visualization of rust fungi structures using fluorochrome Uvitex 2B.

BACKGROUND: Histological examination using fluorochromes is one of the standard methods for observation of microorganisms in tissues and other compartments. In the study of fungi, especially those that cannot be cultured in axenic media such as biotrophic fungi, histological examination of processes associated with the fungal growth, differentiation, infection and other cellular functions can lead to the better understanding of host-parasite interactions. Fluorescence microscopy coupled with Fluorochrome Uvitex 2B have been extensively utilized to study rust fungi structures and host-pathogen interactions. In this study, we report development of a rapid staining protocol of the rust fungus Puccinia triticina using fluorochrome Uvitex 2B. The newly developed rapid procedure was compared with a standard staining technique to observe in planta fungal infection structures development during the wheat-Puccinia triticina interaction. RESULTS: While significantly reducing the time for staining, the rapid protocol described here was equally efficient or better compared to standard procedure in detecting fungal infection structures using Uvitex 2B. In the rapid staining procedure, pre-heating of the stain increased efficiency to detect all the infection structures including haustoria with highly reduced background noise from plant tissue. CONCLUSION: This staining process described here is simple and quick. It can be completed in 4 h, which is of 6 times faster than the standard Uvitex 2B staining procedure.

Migration study of optical brighteners from polymer packing materials to jam squeeze and fruit drink by spectrofluorimetry and RP-HPLC methods.

Optical brighteners are commonly used to modify the appearance and to improve polymer properties of packaging. They are not chemically bound to polymers and able to migrate from packaging into the foods. These migrants are potentially harmful to human health. In concern with human safety an approach was made to analyze three optical brighteners such as diphenylbutadiene, Uvitex-OB, benzophenone in commercial fruit juice and jam. The migration level of these optical brighteners from low density poly ethylene packaging into fruit juice and jam was studied. Two optimized and validated analytical techniques such as spectrofluorimetry and high performance liquid chromatography with photo diode array detector used for migration study. Both methods have shown high correlation coefficients (>0.999), over a concentration range of 0.1-3.2 µg/mL, 0.1-1 µg/mL, 0.05-3.2 µg/mL for diphenylbutadiene, Uvitex-OB and benzophenone respectively. The preliminary studies confirm that the low density poly ethylene layer taken for study contained of diphenylbutadiene and the other two were absent. The migration level of diphenylbutadiene was studied at room temperature and different elevated temperature from 30 °C to 60 °C for up to 3 weeks. At room temperature no migration of diphenylbutadiene was observed where as at higher temperature migration could be observed. The maximum quantity of diphenylbutadiene migrated was found to be 0.0462 mg/kg from tetrapak, and 0.0382 mg/kg from jam squeeze after 3 weeks treatment at 60 °C. The migration of diphenylbutadiene was found to be less than allowable concentration during the study period.