Francis Derouin

Value of antigen detection using an enzyme immunoassay in the diagnosis and prediction of invasive aspergillosis in two adult and pediatric hematology units during a 4-year prospective study

Invasive aspergillosis (IA) is a well recognized, life‐threatening infection in neutropenic patients and stem cell transplantation recipients. Early diagnosis is important to achieve the best outcome for these patients; however, definite proof often is difficult to obtain due to counterindicated invasive procedures.

Survival and Prognostic Factors of Invasive Aspergillosis After Allogeneic Bone Marrow Transplantation

To determine prognostic factors for survival in bone marrow transplant recipients with invasive aspergillosis (IA), we retrospectively reviewed 27 IA cases observed in our bone marrow transplantation unit between January 1994 and October 1994. On 30 September 1997, six patients were alive and disease-free. The median survival after IA diagnosis was 36 days. Of eight variables found to be related to survival according to the univariate analysis, graft-versus-host disease (GVHD) status at IA diagnosis (P = .0008) and the cumulative prednisolone dose taken during the week preceding IA diagnosis (CPDlw) (P < .0001) were selected by a backward stepwise Cox regression model. A three-stage classification was established: CPD1w of < or =7 mg/kg (3 of 8 patients died; 60-day survival rate, 88%), CPD1w of >7 mg/kg and no GVHD (9 of 10 patients died; 60-day survival rate, 20%), and CPD1w of >7 mg/kg and active acute grade 2 or more or extensive chronic GVHD (9 of 9 patients died; 30-day survival rate, 0) (P < .0001).

Culture microtitration: a sensitive method for quantifying Leishmania infantum in tissues of infected mice.

We developed a microtitration method to determine the parasite burdens in homogenized organs of mice infected with Leishmania infantum. This method proved more sensitive than direct enumeration of amastigotes in stained organs, was appropriate for describing the kinetics of infection, and can be considered for physiopathological or pharmaceutical experimental studies.

Contribution of the (1→3)-β-d-Glucan Assay for Diagnosis of Invasive Fungal Infections

ABSTRACT Diagnosis of invasive fungal infection (IFI) remains a challenge. A retrospective study was performed on 279 patients at three French university hospitals to evaluate the performance of the (1→3)-β-d-glucan assay (BG assay; Fungitell; Associates of Cape Cod, Inc.) for the diagnosis of IFI. The results of one serum per subject were analyzed for 117 patients who had probable or proven IFI according to the European Organization for Research and Treatment of Cancer criteria (70 invasive pulmonary aspergilloses [IPA], 27 fungal bloodstream infections, and 20 Pneumocystis jiroveci pneumonias), 40 blood donors, and 122 patients who were hospitalized in hematology wards or intensive care units and were at risk for IFI but in whom IFI had not been diagnosed. For the overall IFI diagnosis, the BG assay had 77.8% sensitivity and specificities of 92.5 and 70.5% for blood donors and patients at risk, respectively. The assay was positive in 48 patients with IPA (68%), in 23 with bloodstream infections (85.2%), and in all who had P. jiroveci pneumonias (100%), and the false-positive rate varied depending on the controls used. It allowed a higher rate of detection among IPA patients compared to the galactomannan enzyme-linked immunosorbent assay (ELISA) (48 versus 39 patients, respectively) and among candidemia patients compared to the mannan ELISA (20 versus 11 patients, respectively). This assay therefore appears to be useful in the diagnosis of IFI, particularly for serum analysis of pneumocystosis pneumonia patients, but further studies are needed to evaluate false-positive rates and its future role in IFI diagnosis.

High Seroprevalence of Encephalitozoon Species in Immunocompetent Subjects

Encephalitozoon species are important pathogens in human immunodeficiency virus-infected patients. However, in immunocompetent persons, little is known about Encephalitozoon infections, mainly because of the lack of reliable diagnostic tests. To improve diagnosis, three serologic techniques that use Encephalitozoon intestinalis as antigen were developed: an ELISA, an immunofluorescence technique (IFAT), and a counterimmunoelectrophoresis (CIE) method. The serologic response against E. intestinalis was studied in sera from 300 Dutch blood donors and 276 pregnant French women. For confirmation of specificity, sera from 150 subjects with various infectious and noninfectious diseases were examined. ELISA, IFAT, and CIE were specific for microsporidia infections, and IFAT and CIE were specific for Encephalitozoon infections. High antibody titers against Encephalitozoon organisms were found in 24 (8%) of 300 Dutch blood donors and in 13 (5%) of 276 pregnant French women. The high seroprevalence against Encephalitozoon species in Dutch blood donors and French women suggests that Encephalitozoon infection is common in immunocompetent subjects.

Detection of Microsporidia and Identification of Enterocytozoon bieneusi in Surface Water by Filtration Followed by Specific PCR.

Microsporidiosis due to species from five different genera, Enterocytozoon, Eiiceplialitozoori, Nosema. Vittafoma and Trachipleistophora, has emerged as an opportunistic infection in immunocompromised patients. Up to now, the source of contamination remains unknown, especially for E. bieneusi, the most common species found in humans. As for other parasitic diseases with fecal excretion of infective stages, the presence of parasites in the environment is likely and surface water may represent a possible source of contamination or a vehicle of transmission. We report on the development of a filtration and PCR-based method for detection of microsporidial spores and we demonstrated the presence of E. bieneusi in one sample of surface water from the river Seine. Experimental studies using water spiked with spores obtained from infected stools (E. bieneusi), or tissue cultures inikted with E. cunicrrli, E. hellem and E. intestinalis (kindly provided by E. Canning and T. Van Gool) were performed to assess for efficiency and sensitivity of the filtration and PCR methods. The filtration comprised two sequential steps. The first step consisted in a concentration of the sample. Water was flushed at 4 litershin. through a filtration cartridge wluch retains all particles of more than 1 micron size (Fulflo. M39R10A, Parker Filtration, France) ; then the collected material was eluted from the cartridge into 2 liters of distilled water. In the second step, this eluate was submitted to three sequential filtrations at 200 d m i n . on 180, 60, 11 microns pore-size nylon filters (47 mm of diameter) (Millipore, France) then finally filtered on a 0.4 micron pore-size polycarbonate filter (Millipore). Each filter was examined by PCR and one part of the 0.4 micron filters was examined by light microscopy using Uvitex 2B staining. DNA extraction of the material colIected from the filters was pertormed using guanidine thiocyanate, according to Boom et al. [l], then identification of parasitic DNA was performed by hvo sequential PCR directed against different DNA fragments of the small sirbunit rRNA gene of microsporidia. The first PCR used generic primers designed for detecting human infecting microsporidia [2] for a 35 cycles amplification, then the resulting products were submitted to a second amplification (35 cycles), using primers specific for E. bieneusi [3], E. intestinalis [ 3 ] , E. cuniculi and E. hellem (kindly provided by ARJ Schuitema). The 1265 bp DNA fragments obtained with E. bieneusi specific PCR primers from 4 filters were sequenced (E.S.G.S., Montigny le Bretonneux, France) and analyzed comparatively to previously published sequences of microsporidia (Genbank). For field studies, five 200400 liters samples were collected : one sample of surface water from the river Seine, 15 km down-stream to Paris, three Goni the river Loire, 25 km up-stream to Nantes, and one sample of ground water collected near Paris. Two liters of each sample were directly filtered (without concentration) and the remaining was concentrated then filtered as described above. Experimental studies using spiked distilled water showed that the spores of the different species of microsporidia were retained by the 0.4 micron filter and could be detected by light microscopy and by PCR. When increasing dilutions of spores of E. cuniculi, E. liellem or E. intestinalis were tested, they were no longer detectable by microscopy but generic and specific PCR were positive, allowing the detection of approximatively 1-3 spores in 2 liters of distilled MATERIAL, AND METHODS.

Detection of Cryptosporidium and Identification to the Species Level by Nested PCR and Restriction Fragment Length Polymorphism

ABSTRACT Cryptosporidiosis is an emerging protozoan disease associated with large waterborne outbreaks. Diagnosis relies on microscopic examination of stools, but this method cannot identify the infecting species of Cryptosporidium. We have developed a test based on nested PCR and restriction fragment length polymorphism (RFLP) that offers simple identification of Cryptosporidium hominis, Cryptosporidium parvum, and most other human infective species in stool samples. Purified C. parvum oocysts were used for PCR development. Extracted DNA was amplified by nested PCR targeting a 214-bp fragment of the 18S RNA gene. Enzymatic restriction sites were identified by bioinformatic analysis of all published Cryptosporidium 18S rRNA sequences. Experiments with spiked stool samples gave an estimated PCR detection limit of one oocyst. Specificity was assessed by testing 68 stool samples from patients with microscopically proven cryptosporidiosis and 31 Cryptosporidium-negative stools. Sixty-seven (98.5%) of the 68 stool samples from patients with microscopically proven cryptosporidiosis and 2 of the other stool samples were positive by PCR and could be genotyped. RFLP analysis identified 36 C. hominis, 19 C. parvum, 8 Cryptosporidium meleagridis, and 6 Cryptosporidium felis or Cryptosporidium canis samples. Species determination in 26 PCR-positive cases was in full agreement with DNA sequencing of the 18S rRNA hypervariable region. The excellent sensitivity of PCR, coupled with the accuracy of RFLP for species identification, make this method a suitable tool for routine diagnosis and genotyping of Cryptosporidium in stools.

Risk Factors for Intestinal Microsporidiosis in Patients with Human Immunodeficiency Virus Infection: A Case-Control Study

A prospective unmatched case-control study was conducted to determine risk factors for intestinal microsporidiosis in persons infected with human immunodeficiency virus (HIV) who had < or = 200 CD4 cells/mm3. In multivariate analysis, case-patients (n = 30) were more likely than were control-subjects (n = 56) to have < or = 100 CD4 cells/mm3 (odds ratio [OR], 6.5; 95% confidence interval [CI], 1-42), to report male homosexual preference (OR, 7.6; 95% CI, 1-59.5), and to report swimming in a pool in the previous 12 months (OR, 9.2; 95% CI, 2.1-38.9). In summary, intestinal microsporidiosis in persons with HIV infection and < or = 200/mm3 CD4 cells is associated with male homosexuality and swimming in pools, suggesting fecal-oral transmission, including sexual and waterborne routes.

In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi.

We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 10(5) spores of Encephalitozoon cuniculi. After 3 days of incubation with various concentrations of drugs, parasitic foci were counted in stained cultures. The inhibition of microsporidial growth exceeding 90% with albendazole (0.005 microgram/ml), fumagillin (0.001 microgram/ml), 5-fluorouracil (3 micrograms/ml), and sparfloxacin (30 micrograms/ml) was observed. Chloroquine, pefloxacin, azithromycin, and rifabutin were partially effective, at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since concentrations that inhibited microsporidia were also toxic for fibroblasts. Pyrimethamine, piritrexim, sulfonamides, paromomycin, roxithromycin, atovaquone, and flucytosine were ineffective. Our results confirm that albendazole and fumagillin have marked activity against E. cuniculi and show the antimicrosporidial activity of 5-fluorouracil and sparfloxacin. These data may form the basis for treatment of Encephalitozoon hellem and Septata intestinalis infections and represent an attempt to identify drugs effective against Enterocytozoon bieneusi. Images

Albendazole for Treatment and Prophylaxis of Microsporidiosis Due to Encephalitozoon intestinalis in Patients with AIDS: A Randomized Double-Blind Controlled Trial

A double-blind placebo-controlled trial was conducted to assess the efficacy and safety of albendazole (400 mg twice daily for 3 weeks) for the treatment of Encephalitozoon intestinalis infection in patients with AIDS. Clearance of microsporidia from the intestinal tract was obtained in 4 of 4 patients in the albendazole group versus 0 of 4 in the control group (P = .01, one-sided Fishers exact test) and was associated with significant clinical benefit. All 4 controls subsequently cleared microsporidia following open-labeled albendazole treatment. To investigate the effect of albendazole in preventing relapse, these 8 patients were then randomly assigned to receive either albendazole (400 mg twice daily) or no treatment for the next 12 months. Albendazole significantly delayed the occurrence of relapse (P = .04, one-sided log-rank test). In human immunodeficiency virus-infected patients with E. intestinalis infection, albendazole has parasitologic and clinical efficacy and reduces the risk of relapse.