C. Pinel

Serum markers for allergic bronchopulmonary aspergillosis in cystic fibrosis: State of the art and further challenges

Allergic bronchopulmonary aspergillosis (ABPA), which results from hypersensitivity, primarily to Aspergillus, represents a severe complication in patients suffering from asthma or cystic fibrosis (CF). Since early treatment of ABPA is supposed to prevent long-term damages, ABPA has to be diagnosed promptly. However, this diagnosis is not straightforward due to clinical and radiological features of ABPA overlapping with those of CF. Despite ABPA specific diagnosis criteria proposed by the Cystic Fibrosis Foundation in 2003, making a definitive ABPA diagnosis in CF patients remains a challenge. Recent advances in the immunopathogenesis of ABPA have initiated the development of new serological tests, such as the recently reported detection of specific IgE to recombinant A. fumigatus allergens, or Thymus- and activation-regulated chemokine (TARC / CCL17), both of which are of value in the diagnosis of APBA. We review in this paper the serum markers that can advance ABPA diagnosis in CF patients, ranging from the well known criteria (anti-A. fumigatus IgE, IgG, and precipitins) to the recent biomarkers (IgE towards recombinant A. fumigatus allergens or TARC detection). Taking into account the up-dated physiopathology of ABPA, we discuss their place and their usefulness, especially TARC, to improve early ABPA detection and monitoring in CF patients.

Characteristics and outcomes of chronic pulmonary aspergillosis: a retrospective analysis of a tertiary hospital registry.

Our objective was to investigate characteristics risk factors and outcomes of patients with chronic pulmonary aspergillosis (CPA).

Seven-year surveillance of nosocomial invasive aspergillosis in a French University Hospital

OBJECTIVESnThis study aims at describing the evolution of the epidemiology of invasive aspergillosis (IA) in a French University Hospital focussing on nosocomial cases, in order to assess the efficiency of the environmental preventive measures which were implemented.nnnMETHODSnFrom 2003 to 2009, IA cases were reviewed monthly and classified according to the EORTC/MSG criteria and the origin of contamination.nnnRESULTSnFive proven and 65 probable IA cases were diagnosed. Most of the cases (74.3%) occurred in patients with haematological malignancies. Incidences of IA and nosocomial IA (NIA) were 0.106 and 0.032 cases per 1000 admissions, respectively. All the 21 NIA cases occurred in the absence of air treatment (laminar air flow facilities or Plasmair decontamination units) and/or during construction works. The 3-month and 1-year overall survival rates were 50.6% [38.2-61.7] and 31.1% [20-42.9] respectively, and did not differ according to the origin of contamination.nnnCONCLUSIONnNosocomial IA still accounted for a third of all IA cases diagnosed from 2003 to 2009 and mainly occurred in the absence of environmental protective measures, which were confirmed to be effective when applied. Our results show that extension and/or reinforcement of these measures is needed, especially in the haematology unit and during construction works.

Characteristic and clinical relevance of Candida mannan test in the diagnosis of probable invasive candidiasis.

The gold standard laboratory tests used to diagnose invasive Candida infection (ICI) are based on the in vitro culture of blood or samples from other sterile sites. However, these tests have limited sensitivity (Se) and are generally not diagnostic until late in the infectious process. The Serion Candida mannan kit was evaluated for the diagnosis of ICI at Grenoble University Hospital (France) between 2007 and 2011. The results were then compared with worldwide data published between 1997 and 2011. This retrospective study was based on follow-up from the investigation of 162 patients of whom 91 had proven ICI; 13 had Candida colonization index (CCI) scores ≥0.42, positive mannan tests, with nonconcomitant infections; and 58 had no evidence of Candida infection. Candida albicans, C. glabrata, C. tropicalis, and C. parapsilosis were the etiologic agents in 104 patients. For patients with or without ICI, the 12-week mortality rates were 35/104 (33.7%) and 6/58 (10.3%), respectively. The mannan diagnostic specificity was 51% and Se was 77%. However, in the meta-analysis (n = 1,536), values were 86% and 62%, respectively. Positive mannan test results may appear early (median 6 days) in the development of candidemia and have moderate diagnostic value for ICI, with a negative predictive value of 83%. In patients at risk of ICI with negative candidemia, the combination of Candida mannan test data with a CCI score ≥0.42 may improve the diagnosis of probable ICI.

Western blot detection of IgG anti-Aspergillus fumigatus elastase in sera of patients with aspergillosis

Anti-Aspergillus fumigatus alkaline protease IgG was investigated by Western blot. Specific IgG antibodies were detected in sera of two of nine patients with proven or highly probable invasive aspergillosis and in sera of one of eight patients with proven or highly probable allergic bronchopulmonary aspergillosis. No response was obtained in sera of 23 control patients. The specific but transient IgG response to this induced enzyme does not recommend its use as sole serodiagnostic aid.

The immunodiagnostic value of six serological techniques in hydatidosis

Abstract Six different serological methods were tested for their ability to diagnose human hydatidosis. These were indirect immunofluorescence, indirect hemagglutination, counter immunoelectrophoresis and enzyme-linked immunosorbent assay (ELISA). In all, 298 sera were examined: 151 sera from negative control subjects, 78 sera from patients with proven hydatidosis, and 69 sera from patients with disease not due to hydatidosis. The last group of samples were used to define the techniques specificity. The specificity and sensitivity of the two recent techniques: enzyme-linked immunoelectrodiffusion assay (ELIEDA) and immunoblotting (electrophoretic migration under denatured and reducing conditions of hydatid fluid antigens, blot and immunoenzymatic assay) were tested mainly on doubtful or weakly positive sera from patients with proven hydatidosis and with filariasis. The combination of enzymelinked immunosorbent assay, indirect immunofluorescence and counter immunoelectrophoresis or ELISA and ELIEDA gave the most dependable and reliable serodiagnostic results with our antigens. ELIEDA is the most useful technique if only one can be used. Our preliminary results with ELIEDA confirm the good specificity and sensitivity of this technique and allow, in some cases, an interpretation of false positive values obtained with ELISA and indirect immunofluorescence. This method also confirms, in most cases, the disease by revealing the arc 5 in patients with low specific IgG content. Immunoblotting in our experimental conditions has a lower sensitivity than ELIEDA but has a specificity for certain proteins (10–12, 15–18, 220, 230 kDa).

Comparative proteomic profiles of Aspergillus fumigatus and Aspergillus lentulus strains by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS)

BackgroundSurface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was applied to analyze the protein profiles in both somatic and metabolic extracts of Aspergillus species. The study was carried out on some Aspergillus species within the Fumigati section (Aspergillus fumigatus wild-types and natural abnormally pigmented mutants, and Aspergillus lentulus). The aim was to validate whether mass spectrometry protein profiles can be used as specific signatures to discriminate different Aspergillus species or even mutants within the same species.ResultsThe growth conditions and the SELDI-TOF parameters were determined to generate characteristic protein profiles of somatic and metabolic extracts of Aspergillus fumigatus strains using five different ProteinChips®, eight growth conditions combining two temperatures, two media and two oxygenation conditions. Nine strains were investigated: three wild-types and four natural abnormally pigmented mutant strains of A. fumigatus and two strains of A. lentulus. A total of 242 fungal extracts were prepared. The spectra obtained are protein signatures linked to the physiological states of fungal strains depending on culture conditions. The best resolutions were obtained using the chromatographic surfaces CM10, NP20 and H50 with fractions of fungi grown on modified Sabouraud medium at 37°C in static condition. Under these conditions, the SELDI-TOF analysis allowed A. fumigatus and A. lentulus strains to be grouped into distinct clusters.ConclusionsSELDI-TOF analysis distinguishes A. fumigatus from A. lentulus strains and moreover, permits separate clusters of natural abnormally pigmented A. fumigatus strains to be obtained. In addition, this methodology allowed us to point out fungal components specifically produced by a wild-type strain or natural mutants. It offers attractive potential for further studies of the Aspergillus biology or pathogenesis.

Does hydrocortisone modify thein vitrosusceptibility ofAspergillus fumigatusto itraconazole and amphotericin B

To analyse if hydrocortisone could modify the in vitro susceptibility of Aspergillus fumigatus to antifungal drugs, we developed a procedure to test the susceptibility of an A. fumigatus strain to amphotericin B and itraconazole, grown in the presence and in the absence of hydrocortisone. Conidia were germinated in the presence or the absence of hydrocortisone in Czapek medium without antifungal drug. A dilution of these cultures (5x10(3) conidia ml(-1)) was spread onto Czapek-agarose plates containing both antifungal drug and hydrocortisone. The cfu per plate were enumerated and compared. A therapeutic concentration of hydrocortisone induced a significant increase in the susceptibility to itraconazole. Conversely, the susceptibility to amphotericin B was not significantly modified when this antifungal drug was associated with hydrocortisone.

IgG1 anti-cell wall and IgG2 anti-phosphopeptidomannan antibodies in the diagnosis of invasive candidiasis and heavy Candida colonization

We conducted a retrospective study to evaluate the usefulness of immunoglobulin G (IgG) subclasses against Candida cell wall fragments (CW) and phosphopeptidomannan (PPM) for the diagnosis of invasive candidiasis (IC). We analyzed 54 patients with IC (n = 19), Candida heavy colonization (HC; n = 16), and controls (no IC or HC, n = 19).In nonneutropenic patients (n = 47), the sensitivity and specificity values of IgG1 anti-CW and IgG2 anti-PPM in IC were 88%, 59%, and 88%, 94%, respectively. The areas under the receiver operating characteristic curves were 0.69 (0.51-0.88) and 0.901 (0.78-1.02), respectively. IgG1 mean values (arbitrary units) and 95% confidence interval were 46 (20-71), 42 (-0.38 to 84) and 20 (8.3-32) in IC, HC, and in controls, respectively, and discriminated IC but not HC from controls (P = .032, and P = .77, respectively). IgG2 mean values were 26 (9.2-42), 19 (4.4-33), and 3.2 (0.28-6.6) in IC, HC, and in controls, respectively, and discriminated both IC and HC from controls (P < .0001 and P = .035, respectively) but did not separate IC from HC (P = .2). IgG2 showed positivity as early as one day after the IC diagnosis. Antibodies were detected in only two out of a total of seven neutropenic patients.For both IC and HC patients, the diagnostic performance of IgG2 anti-PPM was better than the one of IgG1 anti-CW. In nonneutropenic patients, IgG2 anti-PPM accurately identified not only IC patients but also HC patients at high risk for IC. This marker may help clinicians in the initiation of early preemptive therapy.

Chronic Aspergillus fumigatus colonization of respiratory tract in Cystic Fibrosis: Diagnosis, management and antifungal resistance in a French cohort of CF patients

Objectives: To investigate the prevalence of extended-spectrum b-lactamase (ESBL) in Gram-negative bacteria obtained from various clinical specimens of inpatients (from various departments, n = 247) and outpatients (n = 265) in Cantonal Hospital Zenica, Bosnia and Herzegovina during 2009. Methods: Double-disk synergy test was used to detect ESBLs. Minimum inihibitory concentrations (MICs) were determined by broth microdilution method according to CLSI. The transferability of ceftazidime resistance was tested by conjugation (broth mating method). PCR was used to detect alleles encoding ESBL enzymes. Results: ESBL was detected in 168 (68%) and 246 (93%) inand outpatient samples, respectively. ESBL prevalence was highest in nonsurgical departments, 59 (35%, p = 0.000). Both inand outpatient ESBLs were mostly isolated from urine and surgical wounds, 29% and 78%, 27% and 14%, respectively. ESBL/non-ESBL prevalence was highest in an inpatient Klebsiella spp. and in outpatient Escherichia coli isolates, 36%/41% and 34%/42%, respectively. Inpatient urinary isolates were more resistant to all antibiotic tested than outpatient ones. Inpatient surgical wound isolates were more resistant to all cephalosporins than outpatient ones, but lower to other antibiotics. Cefuroxime, ceftazidime and cefotaxime were the least potent antibiotics with MIC90 of 256mg/L. Forty nine (33.6%) of all isolates had plasmid-mediated ampC ESBL by phenylboronic acid phenotypic test. Conjugation frequency was in the range 10−4−10−7. Resistance to gentamicin, chloramphenicol, sulphamethoxazole, trimethoprim and tetracycline in most cases were co transferred alongside with cefotaxime resistance. Thirty four of inpatient urine isolates yielded amplicons with primers specific for TEM, and 33 were positive for CTX-M ESBL. Multiplex PCR revealed group 1 of CTX-M b-lactamases. PCR reactions with SHV-specific primers were positive for K. pneumoniae indicating the presence of intrinsic SHV-1 ESBL. Twenty-one strains had both, CTX-M combined with TEM type of ESBL. Conclusion: The study demonstrated high prevalence of CTX-M b-lactamase in an inpatient urinary isolates associated with high level of resistance to cefotaxime and ceftriaxone. High resistance rates observed for gentamicin and ciprofloxacin probably due to the fact that plasmids encoding ESBLs also contain resistance genes for non b-lactam antibiotics.Objectives: There is a paucity of studies on the genotypic characterisation of invasive S. aureus strains and the incidence of communityacquired methicillin resistant S. aureus (CA-MRSA) infections in South Africa. In this study we characterized S. aureus isolates from bacteraemia episodes using molecular methods and prospectively collected demographic and clinical data on these patients. Methods: Consecutive non-duplicate S. aureus blood culture isolates were prospectively collected over one year. A multiplex PCR was used for the detection of the spa, mecA and pvl genes. The spa gene was sequenced and Ridom StaphType® used to determine spa-types and spa clonal complexes (spa-CC). All cases were categorised by clinical data as either hospital acquired (HA), health-care associated (HCA) or community acquired (CA) S. aureus infections. Data were analysed by using the Statistica® χ2test. A p value less than 0.05 was considered to be statistically significant. Results: 113 S. aureus isolates (70% MSSA, 30% MRSA) were collected from 104 patients. 86 bacteraemia episodes were classified as HA (58%), HCA (27%) and CA (15%). According to clinical data, all CA infections were due to MSSA and no CA-MRSA was detected in our study. In the MSSA subgroup, 45% of cases were classified as HA, 33% HCA and 22% CA. Furthermore, all PVL-positive isolates were MSSA (22.7% of all MSSA). MRSA strains clustered mainly in CC701 and CC012, whereas CC002 only consisted of MSSA (p = 0.0016). The predominant source for S. aureus bacteraemia was catheter-related sepsis (39%). Skin and soft tissue infections and pneumonia were predominantly associated with MSSA strains. Conclusion: Approximately one third of S. aureus bloodstream isolates were MRSA in our setting, a rate comparable to the University Hospital of Wurzburg, Germany. None of the isolates were clinically categorised as CA-MRSA; the majority of isolates were derived from cases defined as HA and the major source was catheter-related sepsis. This information is useful for more targeted infection control and prevention practices to reduce S. aureus bacteraemia.