Invasive aspergillosis and allogeneic hematopoietic stem cell transplantation in children: a 15-year experience.

Despite progress in diagnosis and treatment, invasive aspergillosis (IA) remains a principal cause of mortality due to infection after allogeneic hematopoietic stem cell transplantation (AHSCT). In order to clarify the course of IA among children receiving an AHSCT before the advent of new drugs such as voriconazole or caspofungin, we retrospectively reviewed the medical records of all proven and probable IA between January 1986 and December 2000. 1) Ten children developed IA after AHSCT, mostly long after transplantation. Overall incidence was 2.7%. Seven of those children experienced 1 or more complications after AHSCT and before IA. Mortality was 90% with a median survival of 23 days (2-90). 2) Five children underwent AHSCT after a previous episode of IA. All patients were treated with systemic antifungal therapy combined with surgery. Median time between IA and AHSCT was 110 days (73-370). Two children were diagnosed with IA relapse after transplantation. One child was cured while the other died of IA and AHSCT complications. AHSCT could be considered even in the setting of previous IA, but established strategies implementing newer less toxic antifungal agents as treatment or prophylaxis in high-risk patients are needed.

In vitro susceptibility testing of Candida and Aspergillus spp. to voriconazole and other antifungal agents using Etest: results of a French multicentre study.

Minimum inhibitory concentrations (MICs) of the antifungal agent voriconazole were determined using the Etest and compared with those of amphotericin B, itraconazole and fluconazole using 1986 clinical isolates of Candida spp. Voriconazole MICs were also compared with those of amphotericin B and itraconazole using 391 clinical isolates of Aspergillus spp. Voriconazole was found to have more potent activity and lower MIC values than amphotericin B, itraconazole and fluconazole against C. albicans, C. tropicalis, C. parapsilosis and C. kefyr. Against C. glabrata and C. krusei, voriconazole was more active than either of the other two azole antifungals but had similar activity to amphotericin B. For species of Aspergillus, MIC values of voriconazole were lower than those of amphotericin B and itraconazole against A. fumigatus and A. flavus, and were similar to those of amphotericin B against A. niger. Against A. terreus, MIC values for voriconazole and itraconazole were similar. A. terreus is known to be resistant to amphotericin B, and this was reflected in higher MIC values compared with those of voriconazole and itraconazole. Voriconazole therefore compares very favourably with other antifungal agents against a large number of clinical isolates of Candida and Aspergillus spp.

Prevalence of intestinal protozoans in French patients infected with HIV.

To assess the prevalence of intestinal protozoans in French HIV-infected patients, stool samples, duodenojejunal biopsies, and/or colorectal biopsies from 81 patients were studied for parasites, viruses, and bacteria. Pathogens were found in 70.6% of AIDS patients with diarrhea or malabsorption. The respective prevalence of protozoa in AIDS patients with diarrhea was Cryptosporidium sp.: 37.3%, Blastocystis hominis: 13.7%, Giardia intestinalis: 5.8%, Isospora belli: 2%, Enterocytozoon bieneusi: 2%. Microsporidia were noted in one patient with severe malabsorption but no diarrhea. Other pathogens included cytomegalovirus in 27.4% and Mycobacterium avium in 5.8%. Patients with identified pathogens were more immunosuppressed and more severely malnourished than those with unexplained diarrhea. Multiple pathogens were found in 13 of 81 patients (16%). Twenty-six of 66 identified pathogens (40%) were diagnosed only on biopsy specimens. Chronic diarrhea in HIV patients could be explained in the vast majority by appropriate gastrointestinal investigations. Cryptosporidia played a major role, while microsporidia appeared to be less common.

Plasma itraconazole concentrations in neutropenic patients after repeated high-dose treatment.

Two different treatments with repeated oral high doses of itraconazole were tested for 10 days in 20 neutropenic patients, 10 receiving 400 mg per day and 10 receiving 600 mg per day. In each group 5 patients were treated for acute leukaemia and 5 patients were recipients of autologous bone-marrow transplantation (ABMT). Itraconazole plasma concentrations were assayed by high-performance liquid chromatography. Statistical analysis disclosed a significant interaction between the dispensed dose and the patient types. The difference between the two doses of itraconazole was greater in the ABMT than in the leukaemia patients. After 10 days at 600 mg per day all the ABMT patients had an itraconazole plasma concentration higher than 250 micrograms/l. Therefore, 600 mg per day seems more efficient to obtain a therapeutic level of itraconazole in ABMT patients but this needs to be confirmed for all the neutropenic patients.

Intestinal microsporidiosis occurring in two renal transplant recipients treated with mycophenolate mofetil.

BACKGROUND: Intestinal microsporidiosis is a major cause of chronic diarrhea and malabsorption in patients with human immunodeficiency virus. Its occurrence in transplant recipients has exceptionally been reported to date. METHODS: We report what we believe are the first two cases of intestinal microsporidiosis in renal transplant recipients. The patients were treated with mycophenolate mofetil. RESULTS: The clinical presentation was chronic diarrhea with massive weight loss. Stool analysis revealed microsporidian spores, identified as Enterocytozoon bieneusi spores by polymerase chain reaction. The onset of this opportunistic infection in these two patients is believed to be secondary to an increase in immunosuppression after azathioprine replacement by mycophenolate mofetil. The withdrawal of mycophenolate mofetil led to clinical recovery. CONCLUSION: The incidence of microsporidiosis will probably increase in transplant recipients treated with powerful immunosuppressants. Therefore, we recommend a systematic search for microsporidian spores in stool specimens in cases of unexplained diarrhea in these patients.

Cryptosporidium parvum sporozoite staining by propidium iodide.

Modified Ziehl-Neelsen (ZN) acid-fast stain is the usual method for detection of Cryptosporidium oocysts in feces. Propidium iodide permitted us to stain free or intra-oocyst sporozoites. With the ZN method only 3-5% of the oocysts purified from three human and one experimentally infected lamb dichromate-preserved feces were stained by carbol fuchsin. These fuchsin-stained oocysts were free of intact sporozoites as identified by propidium iodide staining. Treatment with 10% formalin or 0.5% sodium hypochlorite increased the percentage of acid-fast stained oocysts and thus the sensitivity of acid-fast staining. Treatment with sodium hypochlorite induced intra-oocyst sporozoite alterations as demonstrated by flow cytometric analysis of the oocysts' DNA content. Propidium iodide staining of fixed oocysts is a simple and rapid method to visualize sporozoites and to assess oocyst preservation after different treatments.

A longitudinal study of lung transplant recipients infected with Aspergillus: genetic polymorphism of A fumigatus.

BACKGROUND: Aspergillus infection is a well-known complication of lung transplantation and remains associated with high mortality rates. Molecular typing methods are required to elucidate the complex epidemiology of Aspergillus disease in lung transplant recipients. METHODS: Eight lung transplant recipients from one hospital were followed for A fumigatus colonization or infection. Forty-four sequential isolates from these patients were selected and typed by three molecular methods (random amplified polymorphic DNA, sequence-specific DNA primer and multi-locus enzyme electrophoresis). RESULTS: Sixteen different types were identified of which 14 were specific to 1 patient. A factorial correspondence analysis showed that variability between sequential isolates from a single patient was as high as between isolates from the other patients. Lung transplant recipients presented many different genotypes, reflecting the environmental diversity of A fumigatus. Nevertheless, throughout their follow-up, 2 of the 8 lung transplant recipients harbored a common genotype that was not replaced by others. CONCLUSIONS: These results confirm the important genetic polymorphism of the A fumigatus population. The observed genotypes were not related to the type of Aspergillus disease or anti-fungal treatment used nor to the outcome of the patient. These data confirm that all A fumigatus molecular types present the same pathogenic risk.

Pharmacokinetics of itraconazole oral solution in allogeneic bone marrow transplant patients receiving total body irradiation.

A prospective study of the pharmacokinetics of itraconazole solution was performed in 11 patients who underwent allogeneic BMT (day of BMT = day 0) after a conditioning regimen including total body irradiation (TBI). Itraconazole solution (400 mg once a day) was given 7 days before BMT and continued up to the end of neutropenia unless another antifungal treatment was necessary. Blood samples were collected before itraconazole intake (Cmin) and 4 h later (Cmax) every other day for assays of itraconazole (ITRA) and its active metabolite hydroxy-itraconazole (OH-ITRA). The mean values of Cmin ITRA and OH-ITRA, respectively, were 287 +/- 109 ng/ml and 629 +/- 227 ng/ml at day -1 and 378 +/- 147 ng/ml and 725 +/- 242 ng/ml at day +1. The maximum Cmin values were observed at day +3. Six patients at day -1 (54%) and 8 at day +1 (72%) had satisfactory residual plasma concentrations of at least 250 ng/ml of unchanged ITRA. From day +1 to day +9, eight patients discontinued the itraconazole treatment, five of them had satisfactory plasma residual concentrations at this time. This work shows a good bioavailability of itraconazole oral solution during the early phase after allogeneic BMT, but more data are needed for the late phases.

Routine use of a one minute trehalase and maltase test for the identification of Candida glabrata in four laboratories.

AIMS: To evaluate the rapid identification of Candida glabrata using a one minute trehalase and maltase test in four clinical laboratories. METHOD: The test was evaluated with 944 freshly isolated yeasts comprising 572 C glabrata and 372 non-C glabrata strains. These strains were isolated on one of three differential media-Candida ID, CHROMagar Candida, or Albicans ID2 medium-and all strains were fully identified using standard methods. RESULTS: The trehalase and maltase test allowed the overall identification of 550 of 572 C glabrata strains (sensitivity, 96.2%) and only 11 of 372 isolates of other yeast species yielded a false positive result (specificity, 96.8 %). Sensitivity and specificity were consistent from one laboratory to another. Using Candida ID medium, the rapid trehalase and maltase test showed a sensitivity of 95% and specificity of 96.2%. Using CHROMagar Candida, sensitivity and specificity were 95.6% and 98.1%, respectively. Using Albicans ID2 medium (tested by two laboratories), the sensitivity was 100% and 98.5% and specificity was 98.1% and 98.2%. In 60% of cases, the test could be performed directly from the primary isolation medium, thus reducing the time for identification. CONCLUSION: The rapid trehalase and maltase test was highly reliable for the presumptive identification of C glabrata on primary isolation using three different chromogenic media. Direct recognition of C albicans by means of their characteristic colour on chromogenic media coupled with one minute trehalase maltase testing performed only on suspect colonies of C glabrata allowed for rapid presumptive identification of the two yeast species most commonly encountered in clinical samples.

Amphotericin B resistance of Aspergillus terreus in a murine model of disseminated aspergillosis.

The in-vivo activity of amphotericin B and itraconazole against a clinical isolate of Aspergillus terreus was determined in a murine model of disseminated aspergillosis. MICs of amphotericin B and itraconazole for the strain, determined by an NCCLS-based technique, were 2 microg/ml and 1 microg/ml, respectively. Mice infected intravenously were treated with either itraconazole (50 or 100 mg/kg/day) or amphotericin B 4.5 mg/kg/day for 10 days. Treatment with both doses of itraconazole significantly prolonged the survival rates compared with those for untreated mice. In comparison, mortality rate and median survival time were identical for mice treated with amphotericin B and for mice given no therapy, indicating that the strain was highly resistant to amphotericin B in this model. Analysis of sterol composition showed that the major sterol was ergosterol. This suggests that amphotericin B resistance was not related to a modified sterol profile.

In-vivo itraconazole resistance of Aspergillus fumigatus in systemic murine aspergillosis. EBGA Network. European research group on Biotypes and Genotypes of Aspergillus fumigatus.

An animal model of disseminated aspergillosis was used to test the in-vivo activity of itraconazole against four isolates of Aspergillus fumigatus. Two reference isolates of A. fumigatus known to be resistant to itraconazole in vitro and in vivo were used as control isolates, and two new isolates were tested under the same conditions. For each isolate MICs for itraconazole and amphotericin B were determined by an NCCLS-based method. Mice infected intravenously were treated either with itraconazole 100 mg/ kg/day or amphotericin B 4.5 mg/kg/day for 10 days. Amphotericin B showed good in-vivo activity against all four isolates. For one strain, which had a low in-vitro MIC for itraconazole, in-vivo therapy with itraconazole prolonged the survival of mice and reduced fungal burdens in organs compared with untreated controls. In mice infected with a strain with a high MIC of >16 mg/L, itraconazole neither prolonged survival nor reduced fungal load in organs compared with controls. It is concluded that there is a relationship between MIC and treatment outcome in mice for A. fumigatus infection.

Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis. EBGA Network. European Research Group on Biotype and Genotype of Aspergillus.

The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus.

Combination of three typing methods for the molecular epidemiology of Aspergillus fumigatus infections. European Research Group on Biotype and Genotype of Aspergillus.

This study investigated the source of infection and strain relatedness of Aspergillus fumigatus isolates from bronchial colonisation and invasive aspergillosis (IA) in four transplant patients. Environmental isolates from the patient's home and from the hospital and infecting isolates were obtained for patient A who developed IA. Clinic environmental and colonising isolates were obtained for patient B. Sequential isolates were obtained from various organs from patient C who developed IA and also from patient D who had a bronchitic aspergillosis that developed into IA. Ninety-one A. fumigatus isolates were analysed by three typing methods: multi-locus enzyme electrophoresis (MLEE), random amplified polymorphic DNA (RAPD) and sequence-specific DNA primers (SSDP). The three combined typing methods demonstrated a greater differentiation of isolates than the typing methods used separately or in pairs. This demonstrated the genotypic variability of A. fumigatus and facilitated better epidemiological analysis. Large polymorphisms were demonstrated for each patient isolate between and colonies within various samples. The relatedness of the isolates suggested nosocomially acquired aspergillosis for patient B, but the source of infection for patient A remained unclear. The results suggested at least three multiple infections among the four patients. This study enabled the identification of the source of infection and strain relatedness, which in turn facilitates the development of preventive measures for patient management in the future.

Molecular typing of Aspergillus fumigatus strains by sequence-specific DNA primer (SSDP) analysis.

A PCR typing method has been developed and tested to investigate the polymorphism of clinical strains of Aspergillus fumigatus. Firstly, the DNA fragments from random amplified polymorphic DNA (RAPD) patterns of nine epidemiologically and geographically non-related monosporal strains of A. fumigatus were cloned and sequenced. The pairs of five sequence-specific DNA primers (SSDP), characteristic of the 5' and 3' extremities of the RAPD products, were then used in high stringency PCR to type 43 clinical strains of A. fumigatus from 13 patients, according to the presence or absence of a single amplified band. This original approach, which uses the advantages of PCR, has made it possible to overcome the difficulties resulting from the low stringency amplification. The SSDP analysis of 51 A. fumigatus strains (9 unrelated monosporal strains and 43 clinical strains from 13 patients) can be classed into 22 different types with a high reproducibility and a high level of discrimination (D = 0.96). The results suggest that seven lung transplant patients with necrotizing aspergillosis, bronchitis aspergillosis and bronchial colonization were infected by multiple strain genotypes, whereas three patients with invasive aspergillosis seem to have been infected by a single strain.

Hospital-acquired Aspergillus fumigatus infection: can molecular typing methods identify an environmental source?

Aspergillus fumigatus infection in hospitalized immunocompromised patients often raises suspicion regarding the potential for hospital acquisition. Hospital staff have an important responsibility in implementing preventive measures, especially since the advent of current legislation concerning hospital-acquired infections. There have been high expectations that molecular typing methods might determine the source of Aspergillus fumigatus, a ubiquitous mould. The aim of the present epidemiological study, was therefore, to identify the origin(s) of Aspergillus infection in six well-documented patients. All the clinical strains (N=33), and those from hospital (N=14) and home environments (N=34) were isolated according to a standardized protocol and typed by sequence-specific DNA primer analysis. The results confirmed the huge biodiversity of the A. fumigatus population, and consequently the difficulty in ascertaining a hospital source of the infection, as opposed to infections due to other Aspergillus species less frequently encountered.

Host serum protein levels in cysts of human hydatidosis.

8 proteins (albumin, IgG, IgM, IgA, C3c, C4, orosomucoid and alpha 1 antitrypsin) were determined by laser immunonephelometry in hydatid cyst fluid from cysts and sera from 16 patients. The cystic level of albumin was 34.5 +/- SD 59.1 micrograms/ml (range 3.6-85); of IgG 12.9 +/- 18.7 micrograms/ml (1.9-75); of IgM 10.5 +/- 10.4 micrograms/ml (3-37); and of IgA 7.2 +/- 3.5 micrograms/ml (4-19.7). The 4 other proteins represented a smaller fraction: C3c, 0.9 +/- 0.6 micrograms/ml (0.5-2.5); C4, 1.3 +/- 0.9 micrograms/ml (0.5-3.5); orosomucoid, 2.8 +/- 2.3 micrograms/ml (1.4-9.2); and alpha 1-antitrypsin, 5 +/- 4.5 micrograms/ml (2-19). These 8 host proteins constituted 24.6 +/- 24.5% (2.4-76) of the total hydatid cyst fluid proteins (343.7 +/- 172.1 micrograms/ml, range 180-900). The albumin/IgG ration of 3 +/- 2.8 (0.4-10.8) in hydatid cyst fluid was more variable than that in sera, 2 +/- 0.5 (1.2-2.7).

New perspectives in the chemoprophylaxis of toxoplasmosis.

The usual chemoprophylaxis of toxoplasmosis consists of spiramycin or the combination of pyremethamine-sulfamide. This chemoprophylaxis can be used: 1 - In the pregnant woman: spiramycin avoids or has low fetal damage. In case of maternal contamination after 33 weeks of pregnancy, pyrimethamine-sulfadiazine combination should be used in spite of its potential toxicity. 2 - In the newborn infant, chemoprophylaxis prevents the emergence of retinochoroiditis. 3 - In congenital toxoplasmic retinochoroiditis, systematic repetitive cures of pyrimethamine-sulfamide reduce frequency of recurrences. 4 - In immunocompromised patients, the systematic use of pyrimethamine and sulfadoxine prevents neurotoxoplasmosis, in case of kidney, heart, and allogeneic bone marrow transplantations, and also in case of patients with malignant hemopathy or AIDS.

Effect of medium composition on static and cidal activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against Aspergillus fumigatus: a multicenter study.

The effect of the medium composition on the fungistatic (MIC) and fungicidal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilution, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 microl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.

The effect of isatin on the Echinococcus granulosus cyst in an experimental host.

The effect of isatin on the E. granulosus cyst was studied. NMRI mice, which were infected with E. granulosus of sheep origin, were treated daily with isatin at a dose of 50 mg kg-1 for 18 days. Ultrastructural damage was observed in the treated cysts, including accumulation of lamellar stacks, electron-dense granules, autophagosomes and lipid vesicles. Moreover, a biochemical study showed an inhibition of alkaline phosphatase activity, with a decrease in carbohydrate storage and an increase in acid phosphatase activity. In spite of the short duration of the treatment, the results obtained allowed us to conclude that isatin acts on E. granulosus cysts. This activity appears as a process of degeneration linked to the alkaline phosphatase inhibitory effect of isatin.

[Late cryptococcal meningitis after liver transplantation]. / Cryptococcose neuro-méningée tardive après transplantation hépatique.

We report a case of cryptococcal meningitis, eight years after liver transplantation for primary biliary cirrhosis. Detection of the cryptococcal antigen in serum and cerebrospinal fluid appears to be essential for initial diagnosis and follow-up. Oral fluconazole treatment alone can be effective, when given for a very long period to prevent relapse.