Stéphane Bretagne

Comparison of restriction fragment length polymorphism, microsatellite length polymorphism, and random amplification of polymorphic DNA analyses for fingerprinting Aspergillus fumigatus isolates.

ABSTRACT Aspergillus fumigatus fingerprints generated by random amplification of polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) upon hybridization with repeated DNA sequences, and PCR detection of microsatellite length polymorphism (MLP) were compared among 67 isolates. In contrast to RAPD, RFLP and MLP gave discriminating and significantly concordant genotyping results.

Risk of tuberculosis is higher with anti-tumor necrosis factor monoclonal antibody therapy than with soluble tumor necrosis factor receptor therapy: The three-year prospective French Research Axed on Tolerance of Biotherapies registry

OBJECTIVE Tuberculosis (TB) is associated with anti-tumor necrosis factor (anti-TNF) monoclonal antibody (mAb) therapy, but whether this association is drug-specific remains a concern. Our objective was to describe cases of TB associated with anti-TNF mAb therapy, identify risk factors, and estimate the incidence. METHODS We conducted an incidence study and a case-control analysis to investigate the risk of newly diagnosed TB associated with the use of anti-TNF agents. As part of the French Research Axed on Tolerance of Biotherapies (RATIO) registry, for 3 years we collected cases of TB among French patients receiving anti-TNF mAb therapy for any indication; for each case, 2 patients treated with anti-TNF agents served as control subjects. RESULTS We collected 69 cases of TB in patients treated for rheumatoid arthritis (n = 40), spondylarthritides (n = 18), inflammatory colitis (n = 9), psoriasis (n = 1) and Behçets disease (n = 1) with infliximab (n = 36), adalimumab (n = 28), and etanercept (n = 5). None of the patients had received correct chemoprophylactic treatment. The sex- and age-adjusted incidence rate of TB was 116.7 per 100,000 patient-years. The standardized incidence ratio (SIR) was 12.2 (95% confidence interval [95% CI] 9.7-15.5) and was higher for therapy with infliximab and adalimumab than for therapy with etanercept (SIR 18.6 [95% CI 13.4-25.8] and SIR 29.3 [95% CI 20.3-42.4] versus SIR 1.8 [95% CI 0.7-4.3], respectively). In the case-control analysis, exposure to infliximab or adalimumab versus etanercept was an independent risk factor for TB (odds ratio [OR] 13.3 [95% CI 2.6-69.0] and OR 17.1 [95% CI 3.6-80.6], respectively). Other risk factors were age, the first year of anti-TNF mAb treatment, and being born in an endemic area. CONCLUSION The risk of TB is higher for patients receiving anti-TNF mAb therapy than for those receiving soluble TNF receptor therapy. The increased risk with early anti-TNF treatment and the absence of correct chemoprophylactic treatment favor the reactivation of latent TB.

Empirical versus Preemptive Antifungal Therapy for High-Risk, Febrile, Neutropenic Patients: A Randomized, Controlled Trial

BACKGROUND Empirical antifungal therapy is the standard of care for neutropenic patients with hematological malignancies who remain febrile despite broad-spectrum antibacterial treatment. Recent diagnostic improvements may ensure the early diagnosis of potentially invasive fungal disease. Reserving antifungals for this stage may achieve similar survival rates and reduce treatment toxicity and costs. METHODS In this multicenter, open-label, randomized noninferiority trial, we compared an empirical antifungal strategy with a preemptive one. Empirical treatment was defined as antibacterial treatment of patients who have persistent or recurrent fever. Preemptive treatment was defined as treatment of patients who have clinical, imaging, or galactomannan-antigen-assay evidence suggesting fungal disease. First-line antifungal treatment was amphotericin B deoxycholate (1 mg/kg/day) or liposomal amphotericin (3 mg/kg/day), depending on daily renal function. The primary efficacy outcome was the proportion of patients alive at 14 days after recovery from neutropenia. RESULTS The median duration of neutropenia (neutrophil count, <500 cells/mm3) for the 293 patients enrolled was 18 days (range, 5-69 days). By intention-to-treat analysis, survival was 97.3% with empirical treatment and 95.1% with preemptive treatment. The lower 95% confidence limit for the difference in mortality was -5.9%, which was within the noninferiority margin of -8%. Probable or proven invasive fungal infections were more common among patients who received preemptive treatment than among patients who received empirical treatment (13 of 143 vs. 4 of 150; P < .05), and most infections occurred during induction therapy (12 of 73 patients in the preemptive treatment group vs. 3 of 78 patients in the empirical treatment group were infected during induction therapy; P < .01). Preemptive treatment did not decrease nephrotoxicity but decreased costs of antifungal therapy by 35%. CONCLUSIONS Preemptive treatment increased the incidence of invasive fungal disease, without increasing mortality, and decreased the costs of antifungal drugs. Empirical treatment may provide better survival rates for patients receiving induction chemotherapy.

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

BackgroundToxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid.MethodsTwo separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity.ResultsComparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii.ConclusionWe have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

Drug-specific risk of non-tuberculosis opportunistic infections in patients receiving anti-TNF therapy reported to the 3-year prospective French RATIO registry

Background Anti-tumour necrosis factor (TNF) therapy may be associated with opportunistic infections (OIs). Objective To describe the spectrum of non-tuberculosis OIs associated with anti-TNF therapy and identify their risk factors. Methods A 3-year national French registry (RATIO) collected all cases of OI in patients receiving anti-TNF treatment for any indication in France. A case–control study was performed with three controls treated with anti-TNF agents per case, matched for gender and underlying inflammatory disease. Results 45 cases were collected of non-TB OIs in 43 patients receiving infliximab (n=29), adalimumab (n=10) or etanercept (n=4) for rheumatoid arthritis (n=26), spondyloarthritides (n=3), inflammatory colitis (n=8), psoriasis (n=1) or other conditions (n=5). One-third (33%) of OIs were bacterial (4 listeriosis, 4 nocardiosis, 4 atypical mycobacteriosis, 3 non-typhoid salmonellosis), 40% were viral (8 severe herpes zoster, 3 varicella, 3 extensive herpes simplex, 4 disseminated cytomegalovirus infections), 22% were fungal (5 pneumocystosis, 3 invasive aspergillosis, 2 cryptococcosis) and 4% were parasitic (2 leishmaniasis). Ten patients (23%) required admission to the intensive care unit, and four patients (9%) died. Risk factors for OIs were treatment with infliximab (OR=17.6 (95% CI 4.3 - 72.9); p<0.0001)or adalimumab (OR=10.0 (2.3 to 44.4); p=0.002) versus etanercept, and oral steroid use >10 mg/day or intravenous boluses during the previous year (OR=6.3 (2.0 to 20.0); p=0.002). Conclusion Various and severe OIs, especially those with intracellular micro-organisms, may develop in patients receiving anti-TNF treatment. Monoclonal anti-TNF antibody rather than soluble TNF receptor therapy and steroid use >10 mg/day are independently associated with OI.

Recent Exposure to Caspofungin or Fluconazole Influences the Epidemiology of Candidemia: a Prospective Multicenter Study Involving 2,441 Patients

ABSTRACT A prospective multicenter surveillance program on yeast bloodstream infections was implemented in the Paris, France, area without restrictions on ward of hospitalization (intensive care unit, hematology, and surgery) or age (adults and children). The present analysis concerns 2,618 isolates collected over 7 years from 2,441 patients. Centralized species identification and antifungal susceptibility testing using the EUCAST methodology were performed. Almost 10% (232/2,441) of the patients had recently (≤30 days) been treated with antifungal drugs. We analyzed the effect of recent exposure to fluconazole (n = 159) or caspofungin (n = 61) on the proportions of the five major Candida species. For both drugs, preexposure was associated with a decreased prevalence of Candida albicans in favor of less drug-susceptible species (C. glabrata and C. krusei for the former and C. parapsilosis and, to a lesser extent, C. glabrata and C. krusei for the latter; P = 0.001). In the multivariate analysis, the risk of being infected with an isolate with decreased susceptibility to fluconazole was independently associated with an age of ≥15 years (odds ratio [OR] = 2.45; 95% confidence interval [CI] = 1.39 to 4.31; P = 0.002) and with recent exposure to fluconazole (OR = 2.17; 95% CI = 1.51 to 3.13; P < 0.001), while the risk of being infected with an isolate with decreased susceptibility to caspofungin was independently associated with an age <15 years (OR = 2.53; 95% CI = 1.43 to 4.48; P = 0.001) and with recent exposure to caspofungin (OR = 4.79; 95% CI = 2.47 to 9.28; P < 0.001). These findings could influence future recommendations for the management of candidemia.

Aspergillus PCR: one step closer to standardization.

ABSTRACT PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 μl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (≥3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 μl.

Comparison of serum galactomannan antigen detection and competitive polymerase chain reaction for diagnosing invasive aspergillosis.

To improve the diagnosis of invasive aspergillosis (IA), we retrospectively compared competitive polymerase chain reaction (PCR) and sandwich ELISA for detection of serum galactomannan (GM) antigen. We studied 281 serum samples collected weekly during the period at risk for IA from 41 selected hematology patients. Twenty-two patients had confirmed, probable, or suspected IA, according to clinical and mycologic data. Fifteen of them had positive GM titers (87 samples) and 12 had positive PCRs (20 samples). Nineteen of the 20 PCR-positive samples were also GM-positive. Of the 19 patients without IA (83 samples), one had 3 GM-false-positive samples. Neither test anticipated the initiation of antifungal therapy on the basis of clinical suspicion. Both tests were more likely to be positive before death. This study suggests that PCR on serum samples is not more sensitive than GM detection. However, PCR can improve the specificity of the GM test. Together, these noninvasive tests should improve the diagnosis of IA.

Epidemiological trends in invasive aspergillosis in France: the SAIF network (2005–2007)

A prospective (2005-2007) hospital-based multicentre surveillance of EORTC/MSG-proven or probable invasive aspergillosis (IA) cases whatever the underlying diseases was implemented in 12 French academic hospitals. Admissions per hospital and transplantation procedures were obtained. Cox regression models were used to determine risk factors associated with the 12-week overall mortality. With 424 case-patients included, the median incidence/hospital was 0.271/10(3) admissions (range 0.072-0.910) without significant alteration of incidence and seasonality over time. Among the 393 adults (62% men, 56 years (16-84 years)), 15% had proven IA, 78% haematological conditions, and 92.9% had lung involvement. Acute leukaemia (34.6%) and allogeneic stem cell transplantation (21.4%) were major host factors, together with chronic lymphoproliferative disorders (21.6%), which emerged as a new high-risk group. The other risk host factors consisted of solid organ transplantation (8.7%), solid tumours (4.3%), systemic inflammatory diseases (4.6%) and chronic respiratory diseases (2.3%). Serum galactomannan tests were more often positive (≥69%) for acute leukaemia and allogeneic stem cell transplantation than for the others (<42%; p <10(-3)). When positive (n = 245), cultures mainly yielded Aspergillus fumigatus (79.7%). First-line antifungal therapy consisted of voriconazole, caspofungin, lipid formulations of amphotericin, or any combination therapy (52%, 14%, 8% and 19.9%, respectively). Twelve-week overall mortality was 44.8% (95% CI, 39.8-50.0); it was 41% when first-line therapy included voriconazole and 60% otherwise (p <0.001). Independent factors for 12-week mortality were older age, positivity for both culture and galactomannan and central nervous system or pleural involvement, while any strategy containing voriconazole was protective.

ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients.

As culture-based methods for the diagnosis of invasive fungal diseases (IFD) in leukemia and hematopoietic SCT patients have limited performance, non-culture methods are increasingly being used. The third European Conference on Infections in Leukemia (ECIL-3) meeting aimed at establishing evidence-based recommendations for the use of biological tests in adult patients, based on the grading system of the Infectious Diseases Society of America. The following biomarkers were investigated as screening tests: galactomannan (GM) for invasive aspergillosis (IA); β-glucan (BG) for invasive candidiasis (IC) and IA; Cryptococcus Ag for cryptococcosis; mannan (Mn) Ag/anti-mannan (A-Mn) Ab for IC, and PCR for IA. Testing for GM, Cryptococcus Ag and BG are included in the revised EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) consensus definitions for IFD. Strong evidence supports the use of GM in serum (A II), and Cryptococcus Ag in serum and cerebrospinal fluid (CSF) (A II). Evidence is moderate for BG detection in serum (B II), and the combined Mn/A-Mn testing in serum for hepatosplenic candidiasis (B III) and candidemia (C II). No recommendations were formulated for the use of PCR owing to a lack of standardization and clinical validation. Clinical utility of these markers for the early management of IFD should be further assessed in prospective randomized interventional studies.