Mould routine identification in the clinical laboratory by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Background

MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory.

Materials and Methods

First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification.

Results

The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides.

Conclusions

This work''s seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.     

Host defenses trigger salmonella's arsenal

Salmonella survives in macrophages by using a molecular syringe to deliver proteins into the host-cell cytosol where they manipulate phagocyte physiology. Arpaia and colleagues (Arpaia et?al., 2011) show that deployment of this virulence factor is triggered by the very responses that are intended to confer host resistance.     

The effectiveness of a 40-year long iodine prophylaxis in endemic goitre region of Grobnik, Croatia

The region of Grobnik, in the north west of Croatia, 15 km away from the Adriatic coast and 400 meters above the sea level, used to be known as a centre of endemic goitre. Iodine prophylaxis of 10 mg KJ added per kilo salt started in Croatia during the year 1953 and it was increased to 25 mg KJ per kilo in 1996. During 1961, the prevalence of goitre among Grobnik school children was 63%, while in the adult population it was 34%. In 1981, 18% of goitrous school children and 11% of goitrous adults were found in the same region, which shows the fall in goitre prevalence in the twenty-year period, from a severe to a mild one. The aim of this study was to estimate the effectiveness of iodine prophylaxis in goitre eradication and to compare the obtained results to those found in the same region 20 and 40 years ago, namely, in 1961 and 1981. The research was conducted in 2001. We examined 472 Grobnik inhabitants, 378 children (196 girls and 182 boys, aged 7-15 years) and 94 adults. Regarding their size thyroid glands were graded according to WHO and PAHO classification. Data regarding lifestyles and health conditions were collected by individual and family questionnaires. The prevalence of goitre in 2001 was 6.6% in school children and 6.4% in adults. In relation to 1981, we found a statistically significant fall of goitre in school children at the level of p < 0.01 (chi2 = 23.65), but the prevalence change was not statistically significant in adults (p > 0.01, chi2 = 1.419). The frequency of thyroid gland hereditary diseases in native inhabitants was high, 11.7%. There were no statistically significant differences in the prevalence of goitre or thyroid hereditary diseases between groups of native and newcomers' children. According to our results, in the year 2001 the area of Grobnik was still was a region of a mildly expressed endemic goitre. This study presents final results of a 40-year long follow up of endemic goitre eradication, demonstrating the long-term effectiveness of iodine prophylaxis.     

Toward functional genomics of flow-induced outward remodeling of resistance arteries

In resistance-sized arteries, a chronic increase in blood flow leads to increases in arterial structural luminal diameter and arterial wall mass. In this review, we summarize recent evidence that outward remodeling of resistance arteries 1) can help maintain and restore tissue perfusion, 2) is not intimately related to flow-induced vasodilatation, 3) involves transient dedifferentiation and turnover of arterial smooth muscle cells, and 4) is preceded by increased expression of matricellular proteins, which have been shown to promote disassembly of focal adhesion sites. Studies of experimental and physiological resistance artery remodeling involving differential gene expression analyses and the use of knockout and transgenic mouse models can help unravel the mechanisms of outward remodeling.     

Comparison of the Munich Chronotype Questionnaire with the Horne-Ostberg's Morningness-Eveningness Score

We report on results from an Internet survey of sleeping habits in a Dutch population using the Munich Chronotype Questionnaire (MCTQ), supplemented with the Horne-Ostberg Morningness-Eveningness Questionnaire (MEQ). The MCTQ was completed by 5,055 responders, of which 2,481 also completed the MEQ. MEQ score correlated well with the MCTQ assessment of time of mid-sleep on free days (MSF; r = - 0.73) and on workdays (MSW; r = - 0.61). MEQ was more strongly correlated with MSF (50% of sleep time) than with sleep onset (0%), rise time (100%), or with any other percentile (10 to 40, 60% to 90%) of sleep on free days. The study shows that chronotype (based on MSF as measured by the MCTQ) strongly correlates with morningness-eveningness (as measured by the MEQ). However, the MCTQ collects additional detailed information on sleep-wake behavior under natural conditions.     

Development and practical applications of a method for repeated transvaginal, ultrasound-guided biopsy collection of the bovine ovary

In response to the increasing research into primordial and preantral follicular dynamics, a device for transvaginal, ultrasound-guided biopsy collection of the bovine ovary was developed and tested. The new device is based upon a commercially available Ovum Pick-up instrument and consists of a modified needle guidance system, which has been equipped with a trocar needle and caries a 60 cm long true-cut biopsy needle. Biopsies are captured in a 20mm long and 2mm wide specimen notch. In the present experiment, 10 cows were subjected to a twice weekly biopsy regime over a four-week period. A total of 208 attempts at biopsy collection were made, and 141 tissue samples collected (success rate of 68%). Through histological and immunological analyses, these tissue samples have been shown to contain primordial and preantral follicles. At the end of the trial period, several of the donor cows were slaughtered at timed intervals, and the ovaries were harvested for assessment of the damage inflicted by the repeated biopsy procedure. Post mortem ovaries were inspected macroscopically and examined by conventional histological staining. In ovaries retrieved 2 days after the last biopsy session, blood clots were macroscopically apparent throughout the ovaries. Histological examination showed increased infiltration of red blood cells in the ovarian stroma. Analysis from ovaries collected at subsequent slaughter points revealed reduced infiltration of blood, and clear indications of resumed antral follicle development were apparent towards the end of the first month after the trial period. We conclude that the biopsy sampling technique is a repeatable procedure which could serve as a renewable source of primordial and preantral follicles for culture, and as an in vitro model for the study of preantral follicular dynamics.     

Development and application of a brain-specific cDNA microarray for effect evaluation of neuro-active pharmaceuticals in zebrafish (Danio rerio)

The environmental fate and ecotoxicological effect of pharmaceuticals are poorly understood, and standardized tests to detect and evaluate their potential effects in the environment are not available. We developed a zebrafish brain-specific microarray containing 682 neurologically relevant cDNA-fragments. To investigate the applicability of this microarray for studying neurotoxic modes-of-action and impact assessment of neuro-active pharmaceuticals in zebrafish, chlorpromazine was used as a model compound. After exposure to chlorpromazine (75 μg/L) for 2, 4, 14 and 28 days or control treatment RNA was extracted from brains of males and females. Fluorescently labeled cDNA was prepared and hybridized to the custom microarray. In total, 56 genes were differentially expressed in brains of male and/or female zebrafish, of which most genes were down-regulated. A clear difference in response to chlorpromazine exposure between males and females was observed with exposure time as well as in functional classes of affected genes. The presented study is one of the first reports on molecular effects of human neuro-active pharmaceuticals in aquatic non-target organisms. This new genomic tool successfully detected gene expression effects of exposure to chlorpromazine in the brain of zebrafish. Reported gene expression effects are found to be consistent with literature data for other laboratory animals.     

Protein metabolism and strength performance after bovine colostrum supplementation

Summary. This study was designed to determine the responses of muscle protein, serum amino acids, and strength performance to bovine colostrum supplementation in physically active men. The rest (R) group (n=6) and the exercise (E) group (n=6) carried out twice a 2-week experiment randomly assigned in a double-blind fashion with either placebo (PLA; consuming daily 20g maltodextrin) or bovine colostrum (COL; consuming daily 20g colostrum supplement) treatment with one month between. On the test day after the treatment period the measurements were carried out in fasting conditions and E carried out a strength training session (STS). The methods involved the infusion of ring-²H₅-phenylalanine, femoral arterial and venous blood sampling, and biopsies from the vastus lateralis muscle. Serum concentration of essential amino acids during recovery was greater (p<0.05) in the COL groups compared with the PLA groups. Both muscle protein synthesis and breakdown increased (p<0.05) with COL. There were no differences in phenylalanine net balance or strength performance between the PLA and COL groups. It was concluded that a 2-week supplementation with bovine colostrum in physically active men increases serum concentration of essential amino acids but has no effect either on strength performance or protein net balance in fasting conditions during recovery after STS.     

Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males

Loss-of-function mutations of the MECP2 gene at Xq28 are associated with Rett syndrome in females and with syndromic and nonsyndromic forms of mental retardation (MR) in males. By array comparative genomic hybridization (array-CGH), we identified a small duplication at Xq28 in a large family with a severe form of MR associated with progressive spasticity. Screening by real-time quantitation of 17 additional patients with MR who have similar phenotypes revealed three more duplications. The duplications in the four patients vary in size from 0.4 to 0.8 Mb and harbor several genes, which, for each duplication, include the MR-related L1CAM and MECP2 genes. The proximal breakpoints are located within a 250-kb region centromeric of L1CAM, whereas the distal breakpoints are located in a 300-kb interval telomeric of MECP2. The precise size and location of each duplication is different in the four patients. The duplications segregate with the disease in the families, and asymptomatic carrier females show complete skewing of X inactivation. Comparison of the clinical features in these patients and in a previously reported patient enables refinement of the genotype-phenotype correlation and strongly suggests that increased dosage of MECP2 results in the MR phenotype. Our findings demonstrate that, in humans, not only impaired or abolished gene function but also increased MeCP2 dosage causes a distinct phenotype. Moreover, duplication of the MECP2 region occurs frequently in male patients with a severe form of MR, which justifies quantitative screening of MECP2 in this group of patients.