Species Identification and In Vitro Antifungal Susceptibility of Paecilomyces/Purpureocillium Species Isolated from Clinical Respiratory Samples: A Multicenter Study.

Paecilomyces spp. are emerging fungal pathogens, where Paecilomyceslilacinus and Paecilomyces variotii are the most reported species. Taxonomic and phylogenetic revisions in this genus have shown that P. variotii represents a species complex, whereas P. lilacinus is related to another genus called Purpureocillium. The aims of this study were to identify clinical isolates of Paecilomyces spp. at the species level, and to determine their antifungal susceptibility profiles. 70 clinical Paecilomyces spp. isolates were identified by MALDI-TOF Mass Spectrometry (MS) and by multilocus rDNA genes sequencing including ITS and the D1/D2 genes. Among the 70 Paecilomyces spp. isolates, 28 were identified as P. lilacinum, 26 as P. variotii stricto sensu, and 16 as P. maximus. For antifungal susceptibility testing, Minimal Inhibitory Concentrations (MICs) or Minimal Effective Concentrations (MECs) were determined for 8 antifungals. All P. lilacinum isolates had high MICs and MECs of amphotericin B and echinocandins, respectively, unlike P. variotii and P. maximus. For azole drugs, MICs were molecule- and species- dependent. The differences in in vitro susceptibility to antifungals underline the importance of accurate species identification. The MALDI-TOF MS can be a good alternative in routine laboratory to ensure fast identification of Paecilomyces spp. and P. lilacinum.

Invasive aspergillosis due to Aspergillus cryptic species: A prospective multicentre study.

OBJECTIVES: Aspergillus cryptic species are increasingly recognised causes of Aspergillus diseases, including life-threatening invasive aspergillosis (IA). However, as their accurate identification remains challenging in a routine practice, few is known from a clinical and epidemiological perspective. Recently, the MSI application has emerged as a powerful tool for the detection and identification of Aspergillus cryptic species. We aimed to use to the network of users of the MSI application to conduct a multicentre prospective screening of Aspergillus cryptic species-related IA and analyse their epidemiological, clinical and mycological characteristics. METHODS: Over a 27-month period, the clinical involvement of 369 Aspergillus cryptic isolates, from 13 French and Danish MSI application users, was prospectively analysed. Species identification was confirmed by DNA-sequencing and antifungal susceptibility testing was performed using EUCAST reference method. Fifty-one A fumigatus sensu stricto invasive cases were also analysed. RESULTS: Fifteen cryptic isolates were responsible of IA. Eight species were involved, including 5 cases related to the species A sublatus. These species showed high rate of in vitro low susceptibility to antifungal drugs. In comparison with A fumigatus sensu stricto invasive cases, pre-exposure to azole drugs was significantly associated with cryptic IA (P = .02). DISCUSSION: This study brings new insights in cryptic species related IA and underlines the importance to identify accurately at the species level these Aspergillus isolates. The increasing use of antifungal drugs might lead in the future to an epidemiologic shift with an emergence of resistant isolates involved in IA.

Multi-centric evaluation of the online MSI platform for the identification of cryptic and rare species of Aspergillus by MALDI-TOF.

The taxonomy of Aspergillus species has recently been revolutionized with the introduction of cryptic species and section concepts. However, their species-level identification in routine laboratories remains a challenge. The aim of this study was to prospectively assess the identification accuracy of cryptic species of Aspergillus in various laboratories using the mass spectrometry identification (MSI) platform, an independent and freely accessible online mass spectrometry database. Over a 12-month period, when a select set of MSI users identified cryptic species, they were contacted and requested to send the isolates to our laboratory for sequence-based identification. Sequence and MSI identification results were then compared. During the study period, 5108 Aspergillus isolates were identified using MSI including 1477 (28.9%) cryptic species. A total of 245 isolates that corresponded to 56 cryptic species and 13 sections were randomly selected for DNA sequencing confirmation. Agreement between the two methods was 99.6% at the section level and 66.1% at the species level. However, almost all discrepancies (72/83, 86.7%) were misidentifications between closely related cryptic species belonging to the same section. Fifty-one isolates from noncryptic species were also identified, thus yielding 100% and 92.2% agreement at the section and species level, respectively. Although the MSI fungus database is a reliable tool to identify Aspergillus at the section level, the database still requires adjustment to correctly identify rare or cryptic species at the species level. Nevertheless, the application properly differentiated between cryptic and sensu stricto species in the same section, thus alerting on possible specific isolate characteristics.

Evaluation of a New Immunochromatography Technology Test (LDBio Diagnostics) To Detect Toxoplasma IgG and IgM: Comparison with the Routine Architect Technique.

A study comparing the ICT (immunochromatography technology) Toxoplasma IgG and IgM rapid diagnostic test (LDBio Diagnostics, France) with a fully automated system, Architect, was performed on samples from university hospitals of Marseille and Saint-Etienne. A total of 767 prospective sera and 235 selected sera were collected. The panels were selected to test various IgG and IgM parameters. The reference technique, Toxoplasma IgGII Western blot analysis (LDBio Diagnostics), was used to confirm the IgG results, and commercial kits Platelia Toxo IgM (Bio-Rad) and Toxo-ISAgA (bioMérieux) were used in Saint-Etienne and Marseille, respectively, as the IgM reference techniques. Sensitivity and specificity of the ICT and the Architect IgG assays were compared using a prospective panel. Sensitivity was 100% for the ICT test and 92.1% for Architect (cutoff at 1.6 IU/ml). The low-IgG-titer serum results confirmed that ICT sensitivity was superior to that of Architect. Specificity was 98.7% (ICT) and 99.8% (Architect IgG). The ICT test is also useful for detecting IgM without IgG and is both sensitive (100%) and specific (100%), as it can distinguish nonspecific IgM from specific Toxoplasma IgM. In comparison, IgM sensitivity and specificity on Architect are 96.1% and 99.6%, respectively (cutoff at 0.5 arbitrary units [AU]/ml). To conclude, this new test overcomes the limitations of automated screening techniques, which are not sensitive enough for IgG and lack specificity for IgM (rare IgM false-positive cases).

Impact of fluconazole versus posaconazole prophylaxis on the incidence of fungal infections in patients receiving induction chemotherapy for acute myeloid leukemia.

BACKGROUND: Invasive fungal infections (IFIs) remain one of the worrying complications in patients with acute myeloid leukemia (AML) due to their incidence and high level of attributable mortality. In light of these risks, antifungal prophylaxis has always been debated. We conducted a single-center retrospective study of two prophylactic antifungal agents (fluconazole/posaconazole) in 91 consecutive patients receiving induction chemotherapy for AML between 2005 and 2009, in order to evaluate the impact on the incidence of IFI and on the mycological flora of the patients. METHODS: In total, 39 patients received prophylactic fluconazole versus 52 who received posaconazole. The baseline characteristics of the two groups were comparable. RESULTS: Overall, 17 patients developed an IFI, with no difference in frequency between the two groups. Utilization of empirical or pre-emptive therapy was similar irrespective of the type of prophylaxis used. Mycological examination of stools revealed an increase in non-albicans Candida colonization in the fluconazole group during hospitalization and the appearance of Saccharomyces cerevisiae colonization in patients receiving posaconazole. CONCLUSION: The present study does not distinguish between fluconazole and posaconazole as a primary effective prevention against fungal infections. More prospective studies and meta-analyses are warranted.

Severe cutaneous aspergillosis in a premature neonate linked to nonsterile disposable glove contamination?

After having eliminated a dysfunction of the hospital's ventilation system and any other possible environmental reservoir, the investigation of a fatal case of primary cutaneous aspergillosis in a neonate with extremely low birth weight led to the conclusion that nonsterile disposable gloves kept stored in their native packages were the likely source of contamination.

Acquired resistance to voriconazole and itraconazole in a patient with pulmonary aspergilloma.

We describe the development of resistance in an Aspergillus fumigatus strain, originally sensitive to itraconazole and voriconazole, recovered from a case of pulmonary aspergilloma treated with voriconazole. A G448S mutation on the cyp51A gene was detected by sequencing. Frequent culture and in vitro antifungal susceptibility testing is suggested for early detection of the development of multi-azole resistance in patients on long-term therapy for A. fumigatus infections.

Bicentric evaluation of six anti-toxoplasma immunoglobulin G (IgG) automated immunoassays and comparison to the Toxo II IgG Western blot.

A comparative study of the Toxoplasma IgG(I) and IgG(II) Access (Access I and II, respectively; Beckman Coulter Inc.), AxSYM Toxo IgG (AxSYM; Abbott Diagnostics), Vidas Toxo IgG (Vidas; bioMerieux, Marcy l'Etoile, France), Immulite Toxo IgG (Immulite; Siemens Healthcare Diagnostics Inc.), and Modular Toxo IgG (Modular; Roche Diagnostics, Basel, Switzerland) tests was done with 406 consecutive serum samples. The Toxo II IgG Western blot (LDBio, Lyon, France) was used as a reference technique in the case of intertechnique discordance. Of the 406 serum samples tested, the results for 35 were discordant by the different techniques. Using the 175 serum samples with positive results, we evaluated the standardization of the titrations obtained (in IU/ml); the medians (second quartiles) obtained were 9.1 IU/ml for the AxSYM test, 21 IU/ml for the Access I test, 25.7 IU/ml for the Access II test, 32 IU/ml for the Vidas test, 34.6 IU/ml for the Immulite test, and 248 IU/ml for the Modular test. For all the immunoassays tested, the following relative sensitivity and specificity values were found: 89.7 to 100% for the Access II test, 89.7 to 99.6% for the Immulite test, 90.2 to 99.6% for the AxSYM test, 91.4 to 99.6% for the Vidas test, 94.8 to 99.6% for the Access I test, and 98.3 to 98.7% for the Modular test. Among the 406 serum samples, we did not find any false-positive values by two different tests for the same serum sample. Except for the Modular test, which prioritized sensitivity, it appears that the positive cutoff values suggested by the pharmaceutical companies are very high (either for economical or for safety reasons). This led to imperfect sensitivity, a large number of unnecessary serological follow-ups of pregnant women, and difficulty in determining the serological status of immunosuppressed individuals.

Effect of rSAG-1(P30) immunisation on the circulating and tissue parasites in guinea pigs as determined by quantitative PCR.

The efficacy of immunisation with Toxoplasma gondii recombinant protein (rSAG-1) was evaluated in the guinea pig model. For the infectious challenge, two strains, namely, strain C56 (10,000 tachyzoites) and strain 76K (100 cysts), were used to infect a group of 32 guinea pigs each. The circulating, cerebral and pulmonary parasite loads were determined with the real-time polymerase chain reaction (PCR) after immunisation. With the C56 strain, immunisation showed high activity with a reduction of greater than 1 log of the circulating and tissue parasite loads. Thus, there was a significantly lower circulating parasite load in the rSAG-1 + adjuvant group (0.5+/-1.5 Eq parasites/ml) as compared to that in the control group (67+/-110 Eq parasites/ml; p<0.05). In the same manner, the cerebral parasite load was much lower in the rSAG-1 + adjuvant group (10+/-20 Eq parasites/g) than that in the control group (339+/-291 Eq parasites/g; p<0.01). On the other hand, with the 76K strain, the effect of immunisation was much less and that only on the pulmonary parasite load [p(lung)<0.05]. This could be due to the use of different strains and stages of the parasite and/or the difference in the route of administration for challenge. The quantitative PCR technique used has shown a good correlation with animal inoculation, and when associated with the guinea pig model, it seems to be a useful and reproducible technique for future vaccine studies.

Production of antibodies in murine mucosal immunization with Toxoplasma gondii excreted/secreted antigens.

Toxoplasmagondii RH strain excreted/secreted antigens (ESA) were administrated weekly by the oral route, to two groups of 40 OF1 mice for 4 weeks. One group received ESA associated with cholera toxin (CT+) and the other, ESA only (CT-). Five animals from each group were sacrificed from day 4 (D4) to D49 following the first immunization and their feces and sera were collected and tested by ELISA for IgA, IgG and IgM antibody detection. In feces, IgA antibodies were detected on D4 and on D12 in the CT+ and CT- groups, respectively, and they persisted up to D49. IgG antibodies were detected from D12 to D41 in the CT+ group and on D12 only in the CT- group. No IgM antibodies were detected. In sera, IgA antibodies were detected on D27, D41 and D49 only in the CT+ group. IgG and IgM antibodies were found on D12 and D4, respectively, in the CT+ group and starting from D27 in the CT- group. To our knowledge, this is the first demonstration that ESA, with or without CT, are immunogenic when administrated by the oral route.

Reliability of immunoglobulin G antitoxoplasma avidity test and effects of treatment on avidity indexes of infants and pregnant women.

The immunoglobulin G antitoxoplasma avidity test (Vidas; BioMérieux) is an immunoenzymatic test useful for excluding acute infection after the onset of pregnancy. The avidity index (AI) is the ratio of the signal in a test sample washed with urea, which disrupts low-avidity complexes, to that washed without urea. An AI of >0.3 is taken to mean that infection had occurred more than 4 months ago. The increase of the AI with time and the influence of the different treatments given to pregnant women and their newborns were evaluated. A total of 59 pregnant women (271 sera) and their 60 neonates (199 sera) were tested from 1998 to 2002. There were five groups of women based on the type and duration of treatment given. Thirteen pregnant women (group 1) did not receive any treatment, 15 (group 2), 11 (group 3), and 17 (group 4) women received treatment with spiramycin (9 MIU/day) for 0.5 to 2, 2.5 to 5, and 5.5 to 8 months, respectively, and the last 3 women (group 5) received tritherapy (pyrimethamine-sulfonamide and spiramycin alternatively) for 1.5 to 2.5 months. All of the maternal sera collected in the first 6 months had an AI of <0.30, with a mean of 0.07 (range, 0.01 to 0.21). The increase was slow (0.02/month), and there was no significant difference when comparisons were made between the treatment groups. Neonates with proven maternofetal transmission had an increasing AI, unlike those without transmission. However, long-term therapy with pyrimethamine-sulfonamide, as opposed to treatment with spiramycin alone, was found to slow down the progression of the AI. An AI of >0.2 is sufficient to exclude acute infection in pregnant women. In neonates, it is not of major use to diagnose congenital infection; however, it could be a good indicator of compliance and efficacy of treatment of infected infants.

Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens.

Between January 2002 and July 2003, 173 bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100% for staining (where either one or both techniques were positive), 100 and 87.0% for conventional PCR and 100 and 84.9 % for real-time PCR, respectively. The use of a concentration of 10(3) copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84.9 to 98.6% without reducing the sensitivity of the technique. This technique is rapid (<3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with <10(3) copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.