During the past several decades, there has been a steady increase in the frequency of opportunistic invasive fungal infections (IFIs) in immunocompromised patients. However, there is substantial controversy concerning optimal diagnostic criteria for these IFIs. Therefore, members of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group formed a consensus committee to develop standard definitions for IFIs for clinical research. On the basis of a review of literature and an international consensus, a set of research-oriented definitions for the IFIs most often seen and studied in immunocompromised patients with cancer is proposed. Three levels of probability are proposed: "proven," "probable," and "possible." The definitions are intended for use in the context of clinical and/or epidemiological research, not for clinical decision making.

A variety of microorganisms were isolated with frequent mixed infection. NTHi was one of the major pathogens found, especially in association with recurrent CAP, possibly because of improved detection with the FOB with BAL procedure. This suggests that the burden of pediatric CAP could be reduced by addressing NTHi as a major causative pathogen.

This prospective, double-blind trial assessed whether the addition of a glycopeptide would be able to reduce the time to defervescence in neutropenic patients with cancer who had persistent fever 48-60 h after the initiation of empirical piperacillin-tazobactam monotherapy. Of 763 eligible patients, 165 with persistent fever were randomized to receive piperacillin-tazobactam therapy plus either vancomycin therapy or placebo. Defervescence was observed in 82 (95%) of 86 patients in the vancomycin group and in 73 (92%) of 79 patients in the placebo group (P=.52). The distributions of the time to defervescence were not statistically significant between the 2 groups (estimated hazard ratio, 1.03; 95% confidence interval, 0.75-1.43; P=.75). The number of additional episodes of gram-positive bacteremia and the percentage of patients for whom amphotericin B was empirically added to their therapy regimen were also similar in both groups. This study failed to demonstrate that the empirical addition of vancomycin therapy to the treatment regimen is of benefit to persistently febrile neutropenic patients with cancer.

We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.Invasive candidiasis, a common cause of nosocomial infection, is a leading cause of infections among patients receiving bone marrow transplantations or those with leukemia or other cancers and is also associated with a high rate of morbidity and mortality (10,18,22). Given the rapidly fatal course of candidiasis, there is a need for improved methods for early diagnosis and subsequent initiation of antifungal therapy in order to have a significant impact on the death rate (1, 12). One such improvement is the application of rapid and sensitive methods based on PCR that enable the amplification and quantitation of a broad range of fungal pathogens directly from specimens and pure cultures.Two quantitative real-time PCR systems have been described for Candida detection. An assay based on the PCR LightCycler system (Roche Molecular Diagnostics Systems, Indianapolis, Ind.) was applied by Löeffler et al. (15) for quantification of Candida albicans DNA in blood to which known numbers of blastoconidia had been added. Those investigators also used the assay to determine the fungal burdens in a small number of blood samples taken from patients with hematological malignancies. More recently, Guiver et al. (7) and Bowman et al. (3) described the automated detection of fungal DNA by using the TaqMan assay (Applied Biosystems, Foster City, Calif.) with clinical isolates and murine tissues, respect...

Mesenchymal stromal cells (MSCs) are important in the support of hematopoiesis. In this pilot study, we evaluated the safety and efficiency of donor-expanded MSC infusion after allogeneic hematopoietic stem cell transplantation (HSCT) in six patients with poor hematopoietic recovery. MSCs were infused without HSC and without conditioning at a dose of 1 x 10(6)/kg weight. Two patients displayed rapid hematopoietic recovery (days 12 and 21), and four patients showed no response. The two patients who showed hematopoietic recovery were in first complete remission (CR1) compared to the other heavily pretreated patients. There were no toxic side effects linked to MSC infusion. One patient developed cytomegalovirus (CMV) reactivation 12 days following the MSC infusion and died from CMV disease. We found that infusion of MSCs without HSC co-infusion can restore medullary function in some patients with poor hematopoietic recovery. Our data suggest that patients with a less damaged stroma could benefit from this approach.

Molecular diagnosis based on genomic amplification methods such as real-time PCR assay has been reported as an alternative to conventional culture for early detection of invasive candidiasis. However, a major limitation of the molecular method is the difficulty associated with breaking fungal cell walls since the DNA extraction step still requires more than half of a working day. It has been suggested that PCR detection of free template DNA in serum is preferred over the use of whole blood for the diagnosis of systemic candidiasis. In this study, two conventional procedures (the first [the HLGT method] consists of boiling sera in an alkaline guanidine-phenol-Tris reagent, and the second [the PKPC method] uses proteinase K digestion, followed by organic extraction) and three commercially available kits for DNA isolation were evaluated for sensitivity, purity, cost, and use of template for most clinically important Candida species in a TaqMan-based PCR assay. To optimize these procedures, we evaluated the effect of adding 0.5% bovine serum albumin to DNA extracts and found that it decreased the effects of inhibitors. The QIAamp DNA blood kit did significantly shorten the duration of the DNA isolation but was among the most expensive procedures. Furthermore, the QIAamp DNA blood kit proved to be as sensitive as the HLGT DNA isolation method for PCR amplification from 52 serum samples from hematology or oncology patients with clinically proven or suspected systemic Candida infections. All PCR-positive samples showed approximately the same Candida species load by both procedures (100% correspondence), whereas one discordant result was obtained between PCR and blood culture.The management of invasive fungal infections has been hampered by the inability to diagnose the infection at an early stage of disease. However, diagnosis remains difficult, since the only sign of infection may be a prolonged fever that is refractory to antibacterial treatment. In recent years, efforts have been made to develop molecular-biology-based methods for rapid diagnosis, which is crucial to the treatment and recovery of patients suffering from systemic candidiasis. In a comparison of the molecular diagnoses obtained by a real-time PCR-based method to the results of blood culture, the sensitivity and specificity of the molecular method with a C. albicans-specific probe observed with 122 clinical blood samples were 100 and 97%, respectively (12). However, a major limitation of the molecular method in comparison to blood culture was the difficulty associated with problems in breaking fungal cell walls since the DNA extraction step is still a limiting factor, requiring more than half of a working day.Actually, there is no consensus concerning the best blood fractions to be tested for diagnosis of systemic candidiasis. Several PCR methods have been developed for use either on whole-blood samples (7,11,12) or on serum samples (1, 3-5). However, in addition to being too time-consuming and laborintensive, protocols for extraction of cellular candi...

A two-step real-time PCR assay targeting all six capsulation loci of Haemophilus influenzae (i.e., serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131 H. influenzae clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of H. influenzae a to c (10 1 CFU/PCR) was higher than that for type e (10 3 CFU/PCR) and types d and f (10 4 CFU/PCR), and a broader dynamic range was obtained (5 to 8 log 10 units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of H. influenzae invasive infections.

The Progressive Diagnostics Manufacturers epsilometer test (E test; AB Biodisk, Solna, Sweden), a quantitative variant of the disk diffusion technique, was evaluated comparatively to an agar dilution method for the antimicrobial susceptibility testing of Helicobacter pylori. A collection of 79 H. pylori clinical strains, including isolates with known resistance to various antimicrobial agents, was tested against 12 different antimicrobial agents. All strains were tested on Columbia agar supplemented with 10% horse blood. Plates were incubated at 37°C in microaerobic atmosphere (5% 02, 10% C02), and readings were done after 3 days of incubation. In general, E test MICs were easy to interpret and the correlation between MICs by the agar dilution method and the E test was good, with 86 and 99.5% of results being within, respectively, 1 and 2 log2 dilution steps in a total of 936 tests. All strains of H. pylori with documented resistance to the tested agents were detected by the E test. Thus, the E test appears to be an easy and reliable method for determination of MICs of antibiotics for H. pylori, and it may offer an interesting alternative to MIC determination by the agar dilution technique.