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Rapid identification (ID) and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections (BSIs) are essential for the prompt administration of an effective antimicrobial therapy, which can result in clinical and financial benefits. Immediately after blood sampling, empirical antimicrobial therapy, chosen on clinical and epidemiological data, is administered. When ID and AST results are available, the clinician decides whether to continue or streamline the antimicrobial therapy, based on the results of the in vitro antimicrobial susceptibility profile of the pathogen. The aim of the present study is to review and discuss the experimental data, advantages, and drawbacks of recently developed technological advances of culture-based and molecular methods for the diagnosis of BSI (including mass spectrometry, magnetic resonance, PCR-based methods, direct inoculation methods, and peptide nucleic acid fluorescence in situ hybridization), the understanding of which could provide new perspectives to improve and fasten the diagnosis and treatment of septic patients. Although blood culture remains the gold standard to diagnose BSIs, newly developed methods can significantly shorten the turnaround time of reliable microbial ID and AST, thus substantially improving the diagnostic yield.

AimsThe present study was undertaken to evaluate the discrimination ability of six chromogenic media in presumptive yeast identification.MethodsWe analysed 108 clinical isolates and reference strains belonging to eight different species: Candida albicans,Candida dubliniensis, Candida tropicalis, Candida krusei, Candida glabrata, Candida parapsilosis,Candida lusitaniae and Trichosporon mucoides.ResultsC. albicans, C. tropicalis and C. krusei could be distinguished from one another in all the tested chromogenic media, as predicted by the manufacturers. In addition, C. albicans could be distinguished from C. dubliniensis on BBL CHROMagar Candida, Kima CHROMagar Candida and Brilliance Candida, and C. parapsilosis could be identified on CHROMATIC Candida agar, CHROMOGENIC Candida agar, and Brilliance Candida agar.ConclusionsBrilliance Candida provided the widest discrimination ability, being able to discriminate five out of the seven Candida species tested. Interestingly, C. tropicalis and C. krusei could be already distinguished from each other after 24 hours of incubation.

Recent guidelines indicate that oral probiotics, living microorganisms able to confer a health benefit on the host, should be safe for human consumption, when administered in a sufficient amount, and resist acid and bile to exert their beneficial effects (e.g., metabolic, immunomodulatory, anti-inflammatory, competitive). This study evaluated quantitative and qualitative aspects and the viability in simulated gastric and intestinal juices of commercial probiotic formulations available in Italy. Plate counting and MALDI-TOF mass spectrometry were used to enumerate and identify the contained organisms. In vitro studies with two artificial gastric juices and pancreatin–bile salt solution were performed to gain information on the gastric tolerance and bile resistance of the probiotic formulations. Most preparations satisfied the requirements for probiotics and no contaminants were found. Acid resistance and viability in bile were extremely variable depending on the composition of the formulations in terms of contained species and strains. In conclusion, this study indicates good microbiological quality but striking differences in the behavior in the presence of acids and bile for probiotic formulations marketed in Italy.

The spread of multi-drug resistant (MDR) Klebsiella pneumoniae strains producing carbapenemases points to a pressing need for new antibacterial agents. To this end, the in-vitro antibacterial activity of a synthetic N-terminal peptide of human lactoferrin, further referred to as hLF1-11, was evaluated against K. pneumoniae strains harboring different carbapenemase genes (i.e. OXA-48, KPC-2, KPC-3, VIM-1), with different susceptibility to colistin and other antibiotics, alone or in combination with conventional antibiotics (gentamicin, tigecycline, rifampicin, clindamycin, and clarithromycin). An antimicrobial peptide susceptibility assay was used to assess the bactericidal activity of hLF1-11 against the different K. pneumoniae strains tested. The synergistic activity was evaluated by a checkerboard titration method, and the fractional inhibitory concentration (FIC) index was calculated for the various combinations. hLF1-11 was more efficient in killing a K. pneumoniae strain susceptible to most antimicrobials (including colistin) than a colistin-susceptible strain and a colistin-resistant MDR K. pneumoniae strain. In addition, hLF1-11 exhibited a synergistic effect with the tested antibiotics against MDR K. pneumoniae strains. The results of this study indicate that resistance to hLF1-11 and colistin are not strictly associated, and suggest an hLF1-11-induced sensitizing effect of K. pneumoniae to antibiotics, especially to hydrophobic antibiotics, which are normally not effective on Gram-negative bacteria. Altogether, these data indicate that hLF1-11 in combination with antibiotics is a promising candidate to treat infections caused by MDR-K. pneumoniae strains.

The rapid identification and antimicrobial susceptibility testing (AST) of bacteria in clinical blood cultures is crucial to optimise antimicrobial therapy. A previous study involving small sample numbers revealed that the addition of saponin to blood cultures, further referred to as the new method, shortened considerably the turn-around time for the identification and AST of Gram-positive cocci as compared to the current method involving an overnight subculture. Here, we extend previous results and compare the identification and AST of blood cultures containing Gram-negative bacilli by the new and current methods. The identification and AST of 121 Gram-positive and 109 Gram-negative bacteria in clinical monomicrobial blood cultures by the new and current methods and, in the case of Gram-negative bacilli, by direct (no additions) inoculation into an automated system (rapid method) was assessed using the Vitek 2 system. Discrepancies between the results obtained with the different methods were solved by manual methods. The new method correctly identified 88 % of Gram-positive and 98 % of Gram-negative bacteria, and the rapid method correctly identified 94 % of Gram-negative bacteria. The AST for all antimicrobials by the new method were concordant with the current method for 55 % and correct for an additional 9 % of Gram-positive bacteria, and concordant with the current method for 62 % and correct for an additional 21 % of Gram-negative bacilli. The AST by the rapid method was concordant with the current method for 62 % and correct for an additional 12 % of Gram-negative bacilli. Together, saponin-treated monomicrobial blood cultures allow rapid and reliable identification and AST of Gram-positive and Gram-negative bacteria.

ObjectiveFor many years, the treatment of X-linked childhood cerebral adrenoleukodystrophy (XALD) consisted of hydrocortisone replacement and a mixture of short chain-fatty acids, known as ‘Lorenzo's oil’. Recently, bone marrow transplantation (BMT) has also been used.Case reportWe report the case of a patient affected by XALD who developed Graves' hyperthyroidism (GH) and Graves' orbitopathy (GO) after BMT and who we could follow-up for 6.5 years afterwards.Evidence synthesisA boy affected by XALD was treated at the age of 6 years, with a whole BMT from his sister. One year after BMT, the transplanted patient presented TSH at the lower normal value and 3 years later he developed thyrotoxicosis. After a further 2 years, the patient developed GO, which showed clinical evidence of reactivation 5 years after its onset as a consequence of an attempt to treat thyrotoxicosis by means of I131 (300 MBq). Seven years after BMT, the donor showed alterations of thyroid autoimmunity and 1 year thereafter she developed GH. She never presented GO during a subsequent 5 year follow-up.ConclusionsThis case illustrates that autoimmunity originating from a pre-symptomatic donor can be transferred into the host during allogeneic stem cell transplantation. In cases where autoimmune phenomena are recognized in the donor prior to donation, alternative donors or T-cell manipulation of the graft might be considered.

In light of the need for new antifungals, we compared the in vitro antifungal activity of two peptides derived from human lactoferrin (hLF), i.e., hLF(1-11) and hLF(21-31), two analogs of histatin 5, further referred to as dhvar4 and dhvar5, and two ubiquicidin (UBI)-derived peptides, i.e., UBI 18-35 and UBI 29-41, with that of amphotericin B against Aspergillus fumigatus hyphae using the MTT assay. The results revealed a dose-dependent antifungal activity for all peptides, with dhvar5 being the most potent peptide. In addition, hLF(1-11), dhvar5, and UBI 18-35 were effective against A. fumigatus conidia. Furthermore, hLF(1-11) did not lyze human erythrocytes, whereas dhvar5 (>or=16 microM) and UBI 18-35 (>or=20 microM) were hemolytic. Based on these in vitro results and their effectiveness against infections in mice, we concluded that hLF(1-11) and dhvar5 are promising candidates for the development of new agents against A. fumigatus infections.

AIMS: This study aimed to investigate the fate of Bacillus clausii spores orally administered as lyophilized or liquid formulation to healthy volunteers. METHODS AND RESULTS: The study was a randomized, open‐label, cross‐over trial in which two commercial probiotic formulations containing spores of four antibiotic‐resistant B. clausii strains (OC, NR, SIN, T) were given as a single dose administration. Faecal B. clausii units of each strain were counted on selective media and extrapolated for the total weight of evacuated faeces. RAPD‐PCR typing was used to confirm B. clausii identification. Bacillus clausii was found alive in faeces for up to 12 days. In some volunteers, the recovered amount of OC, NR or SIN was higher than the number of administered spores. Bioequivalence among the two formulations was demonstrated. CONCLUSIONS: Bacillus clausii spores survive transit through the human gastrointestinal tract. They can undergo germination, outgrowth and multiplication as vegetative forms. Bacillus clausii strains can have different ability to survive in the intestinal environment. Bacillus clausii spores administered as liquid suspension or lyophilized form behave similarly in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes towards a better understanding of the behaviour of B. clausii spores as probiotics.

Invasive fungal infections are recognized as an important cause of morbidity and mortality in the immunocompromised host. Rapid initiation of adequate antifungal treatment is often hampered by the limitations of current diagnostic methods. This review encompasses the promises and limitations of newer tracers (believed to target the infectious agents), i.e., radiolabeled antimicrobial peptides, antifungals and chitin-specific agents, for fungal infection imaging by scintigraphy. In mice (99m)Tc-labeled peptides derived from human ubiquicidin (UBI29-41) and lactoferrin (hLF1-11) distinguished local Candida albicans and Aspergillus fumigatus infections from sterile inflammatory processes, but not from bacterial infections. Clinical trials showed that (99m)Tc-UBI29-41 can distinguish infections from inflammatory lesions with 80% specificity and 100% sensitivity. (99m)Tc-hLF1-11 was able to monitor the antifungal effects of fluconazole on C. albicans infections. Moreover, (99m)Tc-fluconazole proved to be an excellent tracer for C. albicans infections as it did not accumulate in bacterial infections and inflammatory processes. However this tracer poorly detected A. fumigatus infections. Furthermore, (123)I-chitinase and (99m)Tc-HYNIC-CBP21 accumulated in both C. albicans and A. fumigatus infections in mice at later time points. In conclusion, despite the recent advances in radiolabeled imaging techniques for invasive fungal infections, the search for better tracers for fungal infection imaging should be continued.

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections are essential for the selection of appropriate antimicrobial therapy. To speed up the identification and AST of the causative agent, the fluid from blood culture bottles of a Bactec 9240 instrument (Becton Dickinson) containing Gram-positive cocci was mixed with saponin. After a 15-min incubation, the bacteria were harvested and transferred to the appropriate panel of a BD Phoenix automated microbiology system (Becton Dickinson) for identification and AST. With this approach (referred to as the direct method), we concordantly/correctly identified 56 (82%) of 68 monomicrobial cultures using the results obtained with the method currently used in our laboratory (current method) as comparator. Two (3%) isolates could not be identified and ten (15%) were misidentified. Complete agreement, concerning clinical susceptibility categories and MIC values, between the AST results determined with the direct method and the current method was found for 32 (55%) of 58 isolates. The E-test indicated that the direct method yielded a correct susceptibility profile for 13 of the remaining 26 blood culture isolates. Therefore, a concordant/correct susceptibility profile (with all antimicrobial agents tested) was obtained for 45 (77%) of 58 cultures. The overall error rate amounted to 1.9%, with the majority (1.3%) of errors being minor. Importantly, the results obtained with the direct method were available 12–24 h earlier than those obtained with the current method.

Rapid identification and antimicrobial susceptibility profiling of the bacteria in blood cultures can result in clinical and financial benefits. Addition of saponin to the fluid from blood culture bottles promotes the recovery of the bacteria and thus may shorten the turnaround time of the microbiological analyses. In this study we compared the identification and susceptibility profiles of saponin-treated and untreated (standard method) blood cultures monomicrobial for Gram-positive cocci using Vitek 2. We concordantly identified 49 (89%) of 55 monobacterial cultures using the results with the standard method as reference. Complete categorical agreement between the susceptibility profiles with the new and the standard method was found for 26 (53%) of 49 isolates, while discrepancies were seen for 23 (47%) cultures. E-tests indicated that the new method resulted in a correct susceptibility profile for 8 (35%) of these 23 blood cultures. Therefore, 34 (69%) of 49 cultures showed a concordant/correct susceptibility profile for all antimicrobials with an overall error rate of 2.3%. Thus, addition of saponin to the fluid from blood culture bottles of the Bactec 9240 leads to the rapid (results available a parts per thousand yen12 hours earlier) and reliable identification and susceptibility profiling of Gram-positive cocci in blood cultures with Vitek 2.

The BCG1619c gene of Mycobacterium bovis bacillus Calmette–Guérin (BCG) encodes for a 24 kDa invasin-like protein and is identical to the Rv1566c gene of Mycobacterium tuberculosis . To assess whether this protein was necessary for entry and (or) intracellular persistence in professional phagocytes and (or) in lung epithelial cells, a BCG1619c knockout mutant of M. bovis BCG was generated and compared with the parental BCG strain for its ability to infect and multiply in human monocyte derived THP-1 cells and in the lung epithelial cell line A549. No significant difference between the mutated and the parental BCG strain was observed in either of these in vitro infection systems, indicating that the BCG1619c gene is not essential for cell invasion and intracellular growth of BCG.

BACKGROUND Pulmonary sporotrichosis is a rare event. Sporothrix schenckii is a dimorphic fungus and develops at 37 degrees C in yeast form. Usually hyphae are not observed in tissues, although their presence has been occasionally demonstrated in biopsies. CASE A 37-year-old man, human immunodeficiency virus-1 positive, with a CD4 cell count of 345/mm3, developed a productive cough. A sputum smear revealed the presence of a large amount of long, thin, septated micelia. The hyphae bore oval, sessile conidia. Cultures of sputum yielded numerous colonies of S schenckii. CONCLUSION This is the first report of hyphae of S schenckii in sputum. This case emphasizes the possibilities of cytology for the diagnosis of mycotic infections. Fungi have typical morphologies, and it is possible, on the basis of microscopic evidence, to suspect the nature of the infection early and thus to direct culture procedures.

The aim of this study was to investigate whether technetium-99m labelled fluconazole can distinguish fungal from bacterial infections. Fluconazole was labelled with (99m)Tc and radiochemical analysis showed less than 5% impurities. The labelling solution was injected into animals with experimental infections. For comparison, we used two peptides for infection detection, i.e. UBI 29-41 and hLF 1-11, and human IgG, all labelled with (99m)Tc. Mice were infected with Candida albicans or injected with heat-killed C. albicans or lipopolysaccharides to induce sterile inflammation. Also, mice were infected with Staphylococcus aureus or Klebsiella pneumoniae. Next, accumulation of (99m)Tc-fluconazole and (99m)Tc-labelled peptides/IgG at affected sites was determined scintigraphically. (99m)Tc-fluconazole detected C. albicans infections (T/NT ratio=3.6+/-0.47) without visualising bacterial infections (T/NT ratio=1.3+/-0.04) or sterile inflammatory processes (heat-killed C. albicans: T/NT ratio=1.3+/-0.2; lipopolysaccharide: T/NT ratio=1.4+/-0.1). C. albicans infections were already seen within the first hour after injection of (99m)Tc-fluconazole (T/NT ratio=3.1+/-0.2). A good correlation (R(2)=0.864; P0.05) between T/NT ratios for this tracer and the number of viable C. albicans was found. Although (99m)Tc-UBI 29-41 and (99m)Tc-hLF 1-11 were able to distinguish C. albicans infections from sterile inflammatory processes in mice, these (99m)Tc-labelled peptides did not distinguish these fungal infections from bacterial infections. It is concluded that (99m)Tc-fluconazole distinguishes infections with C. albicans from bacterial infections and sterile inflammations.

The increasing frequency of fungal infections in immunocompromised patients together with the emergence of strains resistant to currently used antifungal drugs point to an increased need for a new class of antimycotics. Antimicrobial peptides are promising candidates for the treatment of fungal infections since they have both mechanisms of action distinct from available antifungal agents and the ability to regulate the host immune defence systems as well. This review focuses on Candida albicans as a large amount of work on the mechanisms of action of classical antifungals as well as antimicrobial peptides, such as defensins, protegrins, histatins and lactoferrin (LF)-derived peptides, has been performed in this yeast. Analogues of these antimicrobial peptides and combinations of antimicrobial peptides with classical antimycotics are under investigation for treatment of candidiasis.

This report describes the use of the 27A probe for the molecular monitoring of Candida albicans infections in liver transplant recipients. Nosocomial candidiasis is the major fungal infection in liver transplant recipients, with Candida albicans being the species most frequently isolated. The molecular epidemiology of Candida albicans infections has been widely investigated, but scant attention has been focused on monitoring the identity of infecting strains in individual patients over the entire course of their hospitalization. In the study presented here, a total of 179 Candida albicans isolates were collected from 10 liver transplant recipients during multiple surveillance cultures performed before and after liver transplantation and from three healthcare workers at the Transplant Unit of Ospedale di Cisanello, Pisa (Italy). Computer-aided analysis of the 27A-probed DNA fingerprints, used to compare the genetic relatedness of all the Candida albicans isolates, showed that most of the patients colonized with Candida albicans before transplantation harbored a unique Candida albicans genotype. This genotype persisted over the entire course of hospitalization and caused multiorgan failure in two patients, both of whom died from endogenously borne Candida albicans infections. Nosocomial acquisition of Candida albicans strains could be monitored in a timely manner in the other patients; for some of them, subsequent strain replacement was registered at different body sites during the post-transplant period. Neither cross-infection between patients nor transmission from healthcare workers to patients occurred in this hospital setting. These results indicate that the molecular monitoring of Candida albicans strains isolated from liver transplant recipients during their hospitalization may provide timely information about the identity of individual Candida albicans strains causing infections.