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This study was undertaken to examine the performance of the Fungitell β-glucan (BG) assay, to compare it with that of the galactomannan (GM) test for the diagnosis of invasive aspergillosis (IA) in patients with hematological malignancies, and to examine the rates of false-positive BG and GM test results due to β-lactam antibiotics among sera of patients with Gram-positive or Gram-negative bacteremia and selected sera with false-positive results from the GM test. Serum samples from 105 patients with proven ( n = 14) or probable ( n = 91) IA, 97 hematology patients at risk for invasive fungal infections, 50 healthy blood donors, and 60 patients with bacteremia were used to study the sensitivities and specificities of the assays. The GM test was more specific than the BG assay (97% versus 82%, respectively; P = 0.0001) and the BG assay was more sensitive than the GM test (81% versus 49%, respectively; P < 0.0001) for IA diagnosis. The study of 49 separate batches of β-lactam antibiotics showed high and very similar rates of false-positive results for the GM and BG assays (29 and 33%, respectively; P = 0.82) but with an almost complete lack of concordance between the 2 assays. For patients with bacteremia, the rate of false-positive results was much higher with the BG test than with the GM test (37% versus 2%, respectively; P < 0.0001), with no significant difference between Gram-positive and Gram-negative bacteremia. In conclusion, the BG test may be useful for the diagnosis of IA because of its high sensitivity in comparison with the GM test, but the overall benefit of this assay remains limited because of its inadequate specificity and its cost.

Invasive aspergillosis (IA) is a major cause of mortality in immunocompromized hosts, most often consecutive to the inhalation of spores of Aspergillus. However, the relationship between Aspergillus concentration in the air and probability of IA is not quantitatively known. In this study, this relationship was examined in a murine model of IA. Immunosuppressed Balb/c mice were exposed for 60 minutes at day 0 to an aerosol of A. fumigatus spores (Af293 strain). At day 10, IA was assessed in mice by quantitative culture of the lungs and galactomannan dosage. Fifteen separate nebulizations with varying spore concentrations were performed. Rates of IA ranged from 0% to 100% according to spore concentrations. The dose-response relationship between probability of infection and spore exposure was approximated using the exponential model and the more flexible beta-Poisson model. Prior distributions of the parameters of the models were proposed then updated with data in a Bayesian framework. Both models yielded close median dose-responses of the posterior distributions for the main parameter of the model, but with different dispersions, either when the exposure dose was the concentration in the nebulized suspension or was the estimated quantity of spores inhaled by a mouse during the experiment. The median quantity of inhaled spores that infected 50% of mice was estimated at 1.8 × 10(4) and 3.2 × 10(4) viable spores in the exponential and beta-Poisson models, respectively. This study provides dose-response parameters for quantitative assessment of the relationship between airborne exposure to the reference A. fumigatus strain and probability of IA in immunocompromized hosts.

Summary The efficacy of antifungal prophylaxis for prevention of invasive aspergillosis (IA) may depend on whether IA results from recent inhalation of spores or reactivation of latent colonisation. Compare the efficacy of liposomal amphotericin B (LAmB) for prophylaxis in acute and reactivation models of IA. In the acute model, mice immunosuppressed from day 0 were challenged at day 3 with an aerosol of Aspergillus fumigatus. LAmB (15 mg kg−1) was administered at day 0 or at challenge. In the reactivation model, naive mice exposed to A. fumigatus remained untreated until clearance of spores from the lungs, then immunosuppressed to induce reactivation. A single LAmB dose was administered at start of immunosuppression. In the acute model, a single administration of LAmB at start of immunosuppression was not effective, but an additional administration resulted in a significant decrease in lung fungal burden (P

International audience; BackgroundAspergillus colonisation is frequently reported after lung transplantation. The question of whether aspergillus colonisation is related to the hospital environment is crucial to prevention.MethodTo elucidate this question, a prospective study of aspergillus colonisation after lung transplantation, along with a mycological survey of the patient environment, was performed.ResultsForty-four consecutive patients were included from the day of lung transplantation and then examined weekly for aspergillus colonisation until hospital discharge. Environmental fungal contamination of each patient was followed weekly via air and surface sampling. Twelve patients (27%) had transient aspergillus colonisation, occurring 1–13 weeks after lung transplantation, without associated manifestation of aspergillosis. Responsible Aspergillus species were A. fumigatus (6), A. niger (3), A. sydowii (1), A. calidoustus (1) and Aspergillus sp. (1). In the environment, contamination by Penicillium and Aspergillus was predominant. Multivariate analysis showed a significant association between occurrence of aspergillus colonisation and fungal contamination of the patient’s room, either by Aspergillus spp. in the air or by A.fumigatus on the floor. Related clinical and environmental isolates were genotyped in 9 cases of aspergillus colonisation. For A. fumigatus (4 cases), two identical microsatellite profiles were found between clinical and environmental isolates collected on distant dates or locations. For other Aspergillus species, isolates were different in 2 cases; in 3 cases of aspergillus colonisation by A. sydowii, A. niger and A. calidoustus, similarity between clinical and environmental internal transcribed spacer and tubulin sequences was >99%.ConclusionTaken together, these results support the hypothesis of environmental risk of hospital acquisition of aspergillus colonisation in lung transplant recipients.

To better understand the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected rats was quantified by means of a real-time polymerase chain reaction assay, in parallel with the kinetics of P. carinii loads in their lungs. P. carinii DNA was detected in the air 1 week after infection and increased until 4-5 weeks after infection before stabilizing. A significant correlation was shown between lung burdens and the corresponding airborne levels, suggesting the possibility of estimating the fungal lung involvement through quantification of Pneumocystis in the exhaled air.

Background. Airborne transmission of Pneumocystis has been demonstrated in animal models and is highly probable in humans. However, information concerning burdens of Pneumocystis jirovecii (human-derived Pneumocystis) in exhaled air from infected patients is lacking. Our objective is to evaluate P. jirovecii air diffusion in patients with Pneumocystis pneumonia. Methods. Patients admitted with Pneumocystis pneumonia were prospectively enrolled from 9 January 2008 to 21 July 2009. Air samples (1.5 m 3 ) were collected on liquid medium with a commercial sampler at 1-, 3-, 5-, and 8-m distances from patients' heads. Air control samples were collected away from Pneumocystis pneumonia patient wards and outdoors. Samples were examined for P. jirovecii detection and quantification using a real-time polymerase chain reaction assay targeting the mitochondrial large subunit ribosomal RNA gene. Results. Forty patients were diagnosed as having Pneumocystis pneumonia. Air sampling was performed in the environment for 19 of them. At a 1-m distance from patients' heads, P. jirovecii DNA was detected in 15 (79.8%) of 19 patients, with fungal burdens ranging from 7.5 X 10 3 to 4.5 X 10 6 gene copies/m 3 . These levels decreased with distance from the patients (P

Toxoplasma gondii can be responsible for congenital toxoplasmosis leading to mild or severe sequelae, and for life-threatening infections in immunocompromised hosts. A new 5'-nuclease real-time PCR assay that targets the 300-fold repeated AF146527 DNA sequence (TaqMan-AF-PCR) has been developed and its performance for diagnosis of toxoplasmosis and treatment follow-up has been assessed. A retrospective analysis was first performed with 144 clinical specimens previously analysed for the presence of T. gondii DNA by a PCR–ELISA assay that targets the B1 gene of T. gondii (B1-PCR–ELISA). Fifteen samples, all from patients with clinically proven toxoplasmosis, were negative according to B1-PCR–ELISA and positive according to TaqMan-AF-PCR. A prospective analysis was then performed with 203 consecutive clinical specimens received at the laboratory of Parasitology of Saint-Louis Hospital during a 4-month period. The diagnosis of toxoplasmosis in two patients was made according to the TaqMan-AF-PCR whereas the B1-PCR–ELISA failed to make diagnosis. Additionally, iterative samples from a patient with cerebral and disseminated toxoplasmosis, already tested using a B1 real-time PCR assay, were tested using the TaqMan-AF-PCR and a Light Cycler real-time PCR assay targeting the same repetitive AF146527 sequence (LC-AF-PCR). Detection was achieved with the TaqMan-AF-PCR, with a mean gain of 7.1 and 3.3 amplification cycles when compared with the B1 real-time PCR and the LC-AF-PCR, respectively. This study demonstrates the higher sensitivity of the 5'-nuclease real-time PCR assay developed for the AF146527 DNA sequence and confirms the interest of using this highly repeated target to improve the diagnosis of toxoplasmosis.

Studies suggest that hospital water supplies may serve as a reservoir for fungi and play a role in their spread. The objective of this study was to provide data on the fungal contamination of water, surfaces, and air of areas of water usage in three hospital wards that admit immunocompromised patients at risk for invasive fungal infection. Air, surface, and water samples were collected in patients’ rooms and water facilities of each ward. Analysis of 215 water samples show differences between wards in fungal recovery rates and isolated species, but Fusarium spp. were the predominant fungi. On the 838 surface samples, a similar fungal diversity was found and Fusarium spp. were the predominant fungi. Aspergillus spp. dominated among the moulds recovered from air. This study confirms that different moulds, mainly Fusarium spp., are present in water and surfaces and that prevention of the risk of nosocomial fungal infection should include a stringent maintenance and cleaning of water facilities.

OBJECTIVE To study the kinetics and identify factors associated with Toxoplasma-specific immune responses in patients with AIDS starting antiretroviral therapy. METHODS A prospective study of 38 HIV-infected patients seropositive for Toxoplasma who started antiretroviral therapy with CD4 T cells less than 200 cells/microl. T-cell and B-cell phenotypes, anti-Toxoplasma antibodies titers, Th-1 and Th-2 cytokine production and lymphocyte proliferative responses (LPRs) to Toxoplasma were assessed over 12 months. RESULTS Median CD4 cell count increased from 122 cells/microl at baseline to 260 cells/microl at 12 months, and the incidence of a positive LPR from 18.4 to 70.5%. A Toxoplasma IgG titer more than 150 IU/ml was the only baseline variable associated with a positive LPR (hazard ratio: 4.6, P = 0.003). Among time-dependent covariates, the number of effector memory (CD45RA-CCR7-) CD4 T cells was associated with a positive LPR (P < 0.02) and the number of terminally differentiated (CD45RA+CCR7-) CD8 T cells was associated with in-vitro production of gamma-IFN (P < 0.008). CONCLUSION Among patients with low CD4 cell counts, high anti-Toxoplasma IgG titers were associated with LPR to Toxoplasma antigen. After starting antiretroviral therapy, the number of effector memory CD4 T cells and terminally differentiated CD8 T cells were associated with the restoration of Toxoplasma LPR and gamma-IFN production, respectively.

Clinical toxoplasmosis in humans has been epidemiologically linked to the consumption of drinking water contaminated by Toxoplasma gondii oocysts. We evaluated killing of T. gondii oocysts after ultraviolet (UV) or ozone treatments by bioassay in mice and/or cell culture. A 4-log inactivation of the oocyst/sporozoite infectivity was obtained for UV fluences >20 mJ cm−2. In contrast, oocysts were not inactivated by ozone with an exposure (Ct) up to 9.4 mg min l−1 in water at 20 °C. In conclusion, UV treatment can be an effective disinfection method to inactivate T. gondii oocysts in drinking water, but ozone did not show promise in this research.

Sulfadiazine, pyrimethamine, and atovaquone are widely used for the treatment of severe toxoplasmosis. Their in vitro activities have been almost exclusively demonstrated on laboratory strains belonging to genotype I. We determined the in vitro activities of these drugs against 17 strains of Toxoplasma gondii belonging to various genotypes and examined the correlations among 50% inhibitory concentrations (IC 50 s), growth kinetics, strain genotypes, and mutations on drug target genes. Growth kinetics were determined in THP-1 cell cultures using real-time PCR. IC 50 s were determined in MRC-5 cell cultures using a T. gondii -specific enzyme-linked immunosorbent assay performed on cultures. Mutations in dihydrofolate reductase (DHFR), dihydropteroate synthase (DHPS), and cytochrome b genes were determined by sequencing. Pyrimethamine IC 50 s ranged between 0.07 and 0.39 mg/liter, with no correlation with the strain genotype but a significant correlation with growth kinetics. Several mutations found on the DHFR gene were not linked to lower susceptibility. Atovaquone IC 50 s were in a narrow range of concentrations (mean, 0.06 ± 0.02 mg/liter); no mutation was found on the cytochrome b gene. IC 50 s for sulfadiazine ranged between 3 and 18.9 mg/liter for 13 strains and were >50 mg/liter for three strains. High IC 50 s were not correlated to strain genotypes or growth kinetics. A new mutation of the DHPS gene was demonstrated in one of these strains. In conclusion, we found variability in the susceptibilities of T. gondii strains to pyrimethamine and atovaquone, with no evidence of drug resistance. A higher variability was found for sulfadiazine, with a possible resistance of three strains. No relationship was found between drug susceptibility and strain genotype.

An experimental protocol was developed to assess the efficacy of two UV reactors (medium-pressure UVaster®, and a low-pressure reactor) on the infectivity of Cryptosporidium parvum oocysts under conditions mimicking small- or medium-size water distribution units. The protocol included purification of large amounts of viable oocysts from experimentally infected calf feces, pilot spiking, sample concentration and purification after UV radiation, oocyst quantification and in vitro evaluation of oocyst infectivity on HCT-8 cells. Water samples were collected at intervals upstream and downstream from the UV reactor after spiking. Oocysts were concentrated by centrifugation, purified by immunomagnetic capture and quantified using laser-scanning cytometry. An enhanced in vitro infectivity test on HCT-8 cells was developed, where oocysts were pretreated in order to obtain maximized in vitro infectivity, and infectious foci were enumerated after immunofluorescence staining after 3 days of culture. This method was superior to viability measured by excystation for assessing oocyst infectivity. The infectivity rate of untreated oocysts ranged between 9% and 30% in replicate experiments. The method allowed us to determine inactivation rates >4.92 (log) with UVaster® and >4.82 with the LP reactor after exposition of oocysts to an effective dose of 400 J m−2 at flow rates of 15 and 42 m3 h−1, respectively.

To date, there is no efficient treatment for cryptosporidiosis and parasite eradication relies on innate and acquired immunity. In this study, we investigated the effect of administration of probiotic bacteria on the development and progression of the experimental infection in suckling rats. Rats were fed daily with 2.10(7) CFU of Lactobacillus casei-containing mixture, starting 2 days before the infection until the spontaneous clearance of the parasite. Effects on weight gain, parasite burden, mucosal histology and production of mucosal cytokines (IFNgamma, IL10 and TNFalpha) were studied. Although a trend to a more rapid clearance of parasites was noted in rats treated with probiotics, no significant effect of probiotics administration was observed in terms of weight gain, parasite burden, mucosal damage, or kinetics of mucosal cytokines during the course of infection. Overall, our results showed that the daily administration of L. casei-containing mixtures was unable to eradicate the parasite in our model.

Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis . A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis -encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis .

A 38-year-old man living near Phnom Penh (Cambodia) was admitted to a hospital in Paris in June 2001 for a single episode of a generalized grand mal seizure. This episode was preceded by a 9-month history of headaches. Magnetic resonance imaging (MRI) of the head revealed a rounded lesion immediately ahead of the left central sulcus. The resected lesion was about 20 mm in diameter. Histologic examination revealed an elongated but unsegmented metacestode at the center of the lesion. Polymerase chain reaction (PCR) analysis was inconclusive due to formalin-based histologic processing of the tissue. Morphologic analysis based on the histologic sections revealed that the metacestode was a tetra-acetabulate plerocercoid of the order Cyclophyllidea, with a distinct rostellum and pseudosegmentation of the dorsoventrally flattened hindbody. This is the first report of a tetra-acetabulate plerocercoid from a human host and the first report of any cyclophyllidean plerocercoid from the human brain. After 6 weeks, the patient was asymptomatic, neurologic examination was normal, and the brain MRI showed only surgical cavitation. The patient returned to Cambodia.

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.

The fungi Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are mainly encountered in (sub)tropical areas as plant pathogens and agents of human dermatomycosis. Because the classification and differentiation of these two species is unclear, we studied 22 S. dimidiatum and 15 S. hyalinum isolates in order to identify potential species-specific insertions and polymorphisms in the 18S subunit ribosomal gene. The presence of an IE intron in S. dimidiatum, together with a single polymorphism (A in S. dimidiatum, G in S. hyalinum) in the coding region, allowed us to differentiate these two species in most cases. Moreover, in one S. dimidiatum isolate we found a group IC1 intron containing a putative truncated His-Cys endonuclease gene. This enzyme shows strong similarity to the intronic homing endonuclease of Physarum polycephalum. Based on these results and our previous findings, we propose an evolutionary pathway for 18S rDNA S. dimidiatum insertions, implying independent events.

The fungi Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are mainly encountered in (sub)tropical areas as plant pathogens and agents of human dermatomycosis. Because the classification and differentiation of these two species is unclear, we studied 22 S. dimidiatum and 15 S. hyalinum isolates in order to identify potential species-specific insertions and polymorphisms in the 18S subunit ribosomal gene. The presence of an IE intron in S. dimidiatum, together with a single polymorphism (A in S. dimidiatum, G in S. hyalinum) in the coding region, allowed us to differentiate these two species in most cases. Moreover, in one S. dimidiatum isolate we found a group IC1 intron containing a putative truncated His-Cys endonuclease gene. This enzyme shows strong similarity to the intronic homing endonuclease of Physarum polycephalum. Based on these results and our previous findings, we propose an evolutionary pathway for 18S rDNA S. dimidiatum insertions, implying independent events.

The in vitro activities of 25 quinolones and fluoroquinolones against erythrocytic stages of Plasmodium falciparum and against liver stages of Plasmodium yoelii yoelii and P. falciparum were studied. All compounds were inhibitory for chloroquine-sensitive and chloroquine-resistant P. falciparum grown in red blood cells. This inhibitory effect increased with prolonged incubation and according to the logarithm of the drug concentration. Grepafloxacin, trovafloxacin, and ciprofloxacin were the most effective drugs, with 50% inhibitory concentrations of P. falciparum and P. yoelii yoelii ; this effect combined reductions of the numbers and the sizes of schizonts in treated cultures. Thus, quinolones have a potential for treatment or prevention of malaria through their unique antiparasitic effect against erythrocytic and hepatic stages of Plasmodium .

A new real-time polymerase chain reaction (PCR) method was developed for quantitation of Enterocytozoon bieneusi DNA in sequential stool specimens from immunocompromised patients with intestinal microsporidiosis. Patients were treated with fumagillin (n=6) or with placebo (n=6), in a randomized comparative trial. At baseline, mean E. bieneusi DNA levels were not significantly different in stool specimens from the placebo group, compared with those from the fumagillin group (5.9+/-0.4 vs. 5.9+/-0.6 log(10) copies/microL of stool suspension, respectively; P=.96). In the placebo group, parasitic burden remained stable during follow-up (P=.46), whereas, in the fumagillin group, E. bieneusi DNA levels dropped below the lower limit of detection in all patients (mean reduction from baseline, -4.7 log(10) copies; P

A new real-time PCR assay for quantitation of Encephalitozoon intestinalis DNA was developed which used a Taq Man fluorescent probe for specific detection. Serial dilutions of E. intestinalis spore suspensions obtained from tissue culture were used as external standards. The detection limit of the technique was 20 spores per ml, with a good interassay reproducibility (coefficient of variation of 7.1% for the suspension containing 20 spores/ml, 5.0% for the suspension containing 75 spores/ml and below 3.5% for higher concentrations). Quantitative detection of E. intestinalis DNA was similar whether the serial dilutions of spores were made in distilled water or in a stool suspension, allowing the use of the assay for stool specimens. The assay was then applied to 14 clinical specimens from 8 immunocompromised patients with proven E. intestinalis infection. The quantitation of the parasitic burden was achieved in stools, blood, urine, tissue biopsies, and bronchopulmonary specimens. The highest parasitic burdens were noted in stools, urine, and bronchopulmonary specimens, reaching 10 5 to 10 6 spores/g or ml. Dissemination of the infection was also evidenced in some patients by demonstration of E. intestinalis DNA in blood and serum. We conclude that real-time PCR is a valuable tool for quantitation of E. intestinalis burden in clinical specimens.

Summary Background The use of combination antiretroviral therapy (cART) has dramatically reduced the prevalence of opportunistic infections, however data on the prevalence of intestinal parasitic infections in HIV-infected patients with low CD4 cell counts in the cART era are scarce. Methods We performed a prospective cohort study among HIV-infected patients with CD4 cell counts 3 seen at a university hospital in Paris. Medical records were reviewed and stool samples were obtained for macroscopic examination and detection of parasites including cryptosporidia and microsporidia, whether or not the patient had diarrhea. Stool cultures were performed for patients with diarrhea. Factors associated with the detection of parasites were then identified. Results Stools samples from 143 consecutive patients were analyzed. Patients were mostly men (76%), and the median patient age was 41 years. The median CD4 cell count was 32/mm 3 , and 59% were receiving cART. Diarrhea was present in 85 patients (59%), 19 of whom (22%) had intestinal parasites detected in stools. Three patients with diarrhea were diagnosed with Salmonella typhimurium , Campylobacter coli , and Clostridium difficile infections. Among the 58 patients without diarrhea, parasitic intestinal pathogens were still identified in six (10%). The overall prevalence of intestinal parasites was 17%, with cryptosporidia ( n =8), microsporidia ( n =6), and Giardia duodenalis ( n =5) being the most frequent pathogens. Patients with intestinal parasites had diarrhea more often (76% vs. 56%, p =0.025) and were more often at US Centers for Disease Control and Prevention (CDC) clinical stage C (84% vs. 69%, p =0.024) than patients without parasites. Conclusions The prevalence of intestinal parasitic infections remains significant in HIV-infected patients with low CD4 counts in the cART era. A systematic search for parasitic pathogens including microsporidia, cryptosporidia, and G. duodenalis should be performed even in the absence of diarrhea.

Intestinal microsporidiosis due to Enterocytozoon bieneusi is a cause of chronic diarrhea, malabsorption, and wasting in immunocompromised patients. Currently, there is no effective treatment.We conducted a randomized, double-blind, placebo-controlled trial of fumagillin (60 mg per day orally for two weeks) in patients with chronic E. bieneusi infection. Efficacy was assessed primarily by the clearance of microsporidia, as evidenced by analysis of stool specimens. Patients in whom microsporidia were not cleared received treatment for two weeks with open-label fumagillin. After clearance of the parasite, follow-up stool examinations were performed monthly to detect relapses.Twelve patients were enrolled in this study, 10 with the acquired immunodeficiency syndrome and 2 who had received organ transplants. Clearance of microsporidia occurred in all six of the patients in the fumagillin group, as compared with none of the six in the placebo group (P=0.002). Treatment with fumagillin was also associated with increases in absorption of D-xylose (P=0.003) and in Karnofsky performance scores (P0.001) and with decreases in loperamide use (P=0.01) and total stool weight (P=0.04). There were serious adverse events (neutropenia and thrombocytopenia) in three patients in the fumagillin group; one patient in the placebo group had severe diarrhea. All six controls subsequently had clearance of microsporidia after open-label treatment with fumagillin. Relapses of the infection were identified in two patients during follow-up (median follow-up, 10 months).Fumagillin is an effective treatment for chronic E. bieneusi infection in immunocompromised patients.

In a multicenter study, potassium dichromate-preserved stools from patients infected with Cryptosporidium parvum ( n = 20), C. hominis ( n = 20), and other Cryptosporidium species ( n = 10) and 60 controls were examined using four immunochromatographic assays. Assay sensitivity ranged between 50.1% and 86.7% for C. parvum and C. hominis but was

The BioPlex 2200 automated analyzer (Bio-Rad Laboratories, Hercules, CA) is a recently developed multiplex analyzer that enables the detection of anti- Toxoplasma , -rubella, and -cytomegalovirus antibodies in the same assay. The aim of this study was to compare this new technology (using the BioPlex 2200 ToRC IgG/IgM kit) in critical cases of serodiagnosis of toxoplasmosis (acute, chronic, or congenital infections and cases with discrepant results) to the technologies used in our routine practice, i.e., the Platelia IgG/IgM enzyme-linked immunosorbent assays (ELISAs) (Bio-Rad Laboratories) and the Toxo-Screen direct agglutination assay (bioMérieux, Lyon, France). Overall, most cases of false-positive/negative results obtained with the Platelia IgG or Toxo-Screen assay were corrected by the BioPlex 2200 ToRC IgG (87.5%). Furthermore, the analysis of 35 sequences of sera showed a trend toward a more rapid decrease of IgM titers by BioPlex 2200 than by Platelia. These results for IgM detection can be explained by a weaker detection of residual IgM. Indeed, among 23 serum samples from patients with probable past infection with long-lasting IgM (Platelia M positive and IgG avidity index, ≥0.5), the BioPlex 2200 Toxoplasma IgM assay was positive for only 11 serum samples. In our panel of critical cases comprising 156 serum and 6 cord blood samples from 103 patients with acute, chronic, or congenital infection, the BioPlex 2200 IgG assay was a sensitive (97.8%) and specific (91.3%) method for IgG detection. The high specificity (97.4%) of IgM detection combined with the shorter kinetics of IgM titers may considerably reduce the number of residual IgM detections, thus yielding more precise diagnoses of acute infections.

HumanLeishmania infantuminfection results in a spectrum of clinical expressions ranging from cutaneous to either asymptomatic or fatal visceral disease. In this context, characterization of parasite virulence appears to be relevant as a biological marker of intrinsic parasitic factors that can affect the pathology of leishmaniasis. Since parasite populations in naturally infected hosts are likely to be composed of multiclonal associations, we first explored the biodiversity of parasite virulence at the intrastrain level in vitro and in vivo by using 11 clones isolated from three strains previously known to express different virulence phenotypes in mice. Subsequently, we studied the course of infection in mice inoculated simultaneously or successively with strains or clones showing various virulence phenotypes. Analysis of in vitro growth characteristics showed no differences among clones from the different parental strains. By contrast, in vivo experiments evidenced a marked intrastrain heterogeneity of virulence to mice. One out of five clones obtained from a virulent strain showed a typical virulence phenotype, while the remaining four clones had low-virulence profiles, as did the six clones isolated from two low-virulence strains. In mixed multiclonal infections, the virulence phenotype was expressed as a dominant character over the associated low-virulence clones. After a challenge with either a homologous or a heterologous strain or clone, virulence phenotypes were conserved and expressed as in naive mice independently from the preexisting population. These results strongly suggest that parasite virulence inL. infantumvisceral leishmaniasis is clonal and dominant in nature.

Invasive aspergillosis (IA) is a major opportunistic infection in haematology patients. Spore inhalation is the usual route of Aspergillus infection, suggesting a determining role of environmental contamination by spores in the epidemiology of IA. We prospectively examined the relationship between environmental contamination by Aspergillus and other fungal species and the incidence of invasive nosocomial aspergillosis (INA) in a bone marrow transplantation unit and two haematology wards. During a four-year period, levels of air and surface fungal contamination were determined bi-monthly in patients' rooms (some equipped with HEPA filters and LAF systems), and various common sites in each ward (corridors, nursing stations, etc.). Results were compared to the incidence of INA. A total of 3100 air and 9800 surface samples were collected, and 79 cases of IA were diagnosed, of which 64 were probably or possibly INA. Patterns of fungal contamination were comparable in the three wards, with a gradient ranging from high levels in common sites to a virtual absence in rooms equipped with HEPA filters and LAF systems. Using a regression model, a significant relationship was found between the incidence of INA and the degree of fungal contamination of air and surfaces in conventional patient rooms (not equipped with HEPA) and common sites. This study shows that in a non-epidemic setting, there is a significant relationship between environmental fungal contamination in haematology wards and the incidence of INA. Our findings underline the importance of environmental surveillance and strict application of preventive measures.

The in vitro lymphocyte proliferative response (LPR) and interferon (IFN)-gamma production in the presence of Toxoplasma antigen were evaluated in 97 human immunodeficiency virus (HIV)-1-infected patients with CD4 cell counts of100 cells/microL (group 1), currently300 cells/microL but previously100 cells/microL (group 2), or always300 cells/microL (group 3) and in 28 non-HIV-infected blood donors (group 4), all seropositive to Toxoplasma. In group 2, 81% of patients had a positive LPR, versus 20% in group 1 (P10(-3)). IFN-gamma production was greater in group 2 than in group 1 (922 vs. 0 Deltapg/mL; P=10(-4)). Multivariate analysis found a significant association between a positive LPR to Toxoplasma antigen and a CD4 count300 cells/microL (odds ratio [OR], 16.3; 95% confidence interval [CI], 5.3-50.2) and anti-Toxoplasma IgG titer150 IU/mL (OR, 5; 95% CI, 1.6-15.2). Immune reconstitution under highly active antiretroviral therapy was associated with a restoration of immune responses against Toxoplasma.

In order to define more accurately human immunodeficiency virus-infected patients at risk of developing toxoplasmic encephalitis (TE), we assessed the prognostic significance of the anti- Toxoplasma gondii immunoglobulin G (IgG) immunoblot profile, in addition to AIDS stage, a CD4 + cell count 3 , and an antibody titer ≥150 IU/ml, in patients with CD4 cell counts 3 and seropositive for T. gondii. Baseline serum samples from 152 patients included in the placebo arm of the ANRS 005-ACTG 154 trial (pyrimethamine versus placebo) were used. The IgG immunoblot profile was determined using a Toxoplasma lysate and read using the Kodak Digital Science 1D image analysis software. Mean follow-up was 15.1 months, and the 1-year incidence of TE was 15.9%. The cumulative probability of TE varied according to the type and number of anti- T. gondii IgG bands and reached 65% at 12 months for patients with IgG bands of 25 and 22 kDa. In a Cox model adjusted for age, gender, Centers for Disease Control and Prevention (CDC) clinical stage, and CD4 and CD8 cell counts, the incidence of TE was higher when the IgG 22-kDa band (hazard ratio [HR] = 5.4; P < 0.001), the IgG 25-kDa band (HR = 4.7; P < 0.001), or the IgG 69-kDa band (HR = 3.4; P < 0.001) was present and was higher for patients at CDC stage C (HR = 4.9; P < 0.001). T. gondii antibody titer and CD4 cell count were not predictive of TE. Thus, detection of IgG bands of 25, 22, and/or 69 kDa may be helpful for deciding when primary prophylaxis for TE should be started or discontinued, especially in the era of highly active antiretroviral therapy.

The aim of this study was to assess the performance of different impactor air samplers for fungal spore collection in the hospital environment. Four recent impactor air samplers were selected: Samplair (AES, Combourg, France); Air Test Omega (LCB, France); Air Samplair Mas-100 (Merck, France); and BioImpactor 100-08 (AES). They were compared with one another at three different hospital sites with varying levels of contaminated air. No significant difference in the efficiency of spore recovery was found between Air Test Omega, Mas-100 and BioImpactor, whereas Samplair was significantly less efficient. BioImpactor was then selected to represent the three superior impactors and was compared with the single-stage Andersen disposable sampler and the Collectron MD8 air sampler (Sartorius, France) and the High Flow Air Sample (BioTest, France), which are based on filtration and centrifugation methods, respectively. No significant difference was observed in terms of spore recovery. On the basis of their performance, unit sampling cost, autonomy and simplicity of use, we conclude that Air Test Omega, Air Samplair Mas-100 and BioImpactor 100-08 are suitable for routine indoor evaluation of fungal contamination of air in hospitals.

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of protozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl-beta-D-ribofuranoside (NPR) substrate for the quantification of Leishmania infantum. The free promastigote stage of L. infantum contains high amounts of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide range of concentrations (5 x 10(4) 2 x 10(8) parasites ml(-1)) in a single assay. No difference in NH activity was observed between various strains at equivalent concentrations and growth curves determined with NH dosage were similar to optical counts. Samples can be stored at -20 degrees C for weeks before use in this unique assay without significant loss of NH activity. The method is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. infantum promastigotes in vitro.

Disseminated microsporidiosis is a life-threatening opportunistic infection. Here, we report about a previously undescribed genovar of Encephalitozoon cuniculi causing disseminated infection in a non-HIV-infected renal transplant recipient. Disseminated microsporidiosis must be considered in the differential diagnosis of chronic fever in renal allograft recipients, even those without urinary symptoms.

C.B-17 SCID and congenic BALB/C mice were used to examine Leishmania infantum strain pathogenicity independently of host genetic factors. While parasite loads were significantly higher in immunodeficient mice than in immunocompetent mice, the kinetics of infection during a long-term follow-up were similar, suggesting that intrinsic parasitic factors also influence the outcome of L. infantum infection.

In order to estimate the rate and seasonal variation of Enterocytozoon bieneusi contamination of surface water, sequential samples of water from the River Seine in France were collected during a 1-year period. Each sample (300-600 l) was submitted to sequential filtrations, and the filters were then examined for microsporidia using light microscopy and nested polymerase chain reaction (PCR) for E. bieneusi. Amplified products were hybridized with a E. bieneusi-specific probe. Twenty-five samples of water were analyzed during 1 year. Microscopic examination of stained filters proved unreliable for the identification of spores. Using nested PCR, 16 of 25 specimens were positive (64%). Unexpectedly, E. bieneusi was identified in only one sample by specific hybridization underlining the lack of specificity of ours primers. Nevertheless, using DNA sequence analysis, unknown microsporidia species were identified in eight cases, which had highest scores of homology with Vittaforma corneae or Pleistophora sp. This study shows a low rate of water contamination by E. bieneusi suggesting that the risk of waterborne transmission to humans is limited.

The apicoplast, a plastid-like organelle of Toxoplasma gondii , is thought to be a unique drug target for quinolones. In this study, we assessed the in vitro activity of quinolones against T. gondii and developed new quantitative structure-activity relationship models able to predict this activity. The anti- Toxoplasma activities of 24 quinolones were examined by means of linear discriminant analysis (LDA) using topological indices as structural descriptors. In parallel, in vitro 50% inhibitory concentrations (IC 50 s) were determined in tissue culture. A multilinear regression (MLR) analysis was then performed to establish a model capable of classifying quinolones by in vitro activity. LDA and MLR analysis were applied to virtual structures to identify the influence of each atom or substituent of the quinolone ring on anti- Toxoplasma activity. LDA predicted that 20 of the 24 quinolones would be active against T. gondii . This was confirmed in vitro for most of the quinolones. Trovafloxacin, grepafloxacin, gatifloxacin, and moxifloxacin were the quinolones most potent against T. gondii , with IC 50 s of 0.4, 2.4, 4.1, and 5.1 mg/liter, respectively. Using MLR analysis, a good correlation was found between measured and predicted IC 50 s ( r 2 = 0.87, cross-validation r 2 = 0.74). MLR analysis showed that the carboxylic group at position C-3 of the quinolone ring was not essential for anti- Toxoplasma activity. In contrast, activity was totally dependent on the presence of a fluorine at position C-6 and was enhanced by the presence of a methyl group at C-5 or an azabicyclohexane at C-7. A nucleophilic substituent at C-8 was essential for the activity of gatifloxacin and moxifloxacin.

Objective: Intestinal microsporidiosis caused by Enterocytozoon bieneusi is a cause of chronic diarrhoea in patients with HIV infection for which there is no current therapy. This study was designed to assess the safety and efficacy of oral fumagillin in this infection. Design: A dose-escalation trial. Methods: Twenty-nine HIV-infected patients with E. bieneusi infection were consecutively enrolled in the trial. Oral doses of fumagillin were given to four groups of patients for 14 days: 10 mg/day (group 1), 20 mg/day (group 2), 40 mg/day (group 3), and 60 mg/day (group 4). Patients were seen at weeks 1, 2, 4 and 6 to assess safety and efficacy. Efficacy was assessed primarily by the the clearance of microsporidia from stools and follow-up duodenal biopsies. Results: Thirteen patients complained of abdominal cramps, vomiting or diarrhoea during the study, and three patients had fumagillin withdrawn because of adverse events. Thrombocytopenia, neutropenia and hyperlipasaemia were the most frequent biological adverse events. Twenty-one out of 29 patients transiently cleared microsporidia from their stools during the study. By week 6, however, all patients in groups 1, 2 and 3 had parasitic relapse. Interestingly, eight out of 11 (72%) patients treated with 60 mg/day (group 4) apparently cleared microsporidia from their gastrointestinal tract and gained weight. No parasitic relapse was documented in these eight patients during a mean follow-up of 11.5 months. Conclusion: Treatment with fumagillin at 60 mg/day for 14 days has promise as an effective oral treatment for E. bieneusi infections.

The aims of the present study were to design an easy and sensitive DNA amplification method for detection of Toxoplasma gondii with low risk of accidental contamination, and to find a rapid method for purification of clinical samples containing potential inhibitors of the amplification reaction. With a pair of primers amplifying a 619-bp fragment of the B1 gene of this parasite it was possible to detect DNA equivalent to 10 parasites. When a third primer was added to the same tube, sensitivity increased to 0.1 parasite. In a comparison of different DNA purification methods, the High Pure PCR Template Preparation Kit (Boehringer Mannheim, Germany) gave the best results. With this purification method and the one-tube hemi-nested PCR, T. gondii DNA was detected in 14 (87.5%) of 16 clinical specimens (amniotic fluid, broncho-alveolar lavage, bone marrow, blood, liver biopsy) in which the parasite was demonstrated by cell culture.

Among 186 suspected cases of Toxoplasma encephalitis (TE) in HIV-infected patients, 113 were classified as TE and 73 as non-TE. Serum Toxoplasma gondii (T.g.) antibodies were detected by ELISA in 97% of TE vs 71% of non-TE cases (p < 0.001). In the 164 patients positive for T. g. antibodies, the IgG 27 and 32 bands were more frequently present in TE than in non-TE (p = 0.003, p = 0.002, respectively). Among patients with positive T.g. serology, multivariate analysis showed that the presence of an IgG32 (OR 3.1) or IgG27 band (OR 2.7) on Western blot was highly indicative of TE independently of each other. Positive T.g. serology, but not anti-T.g. IgG antibody titres, was predictive. Thus, the positivity of IgG 27 and 32 bands on Western blot analysis provides useful data for improving the diagnosis of presumptive TE in HIV-infected patients with suspected TE and positive Toxoplasma serology.

The incidence of cerebral and extracerebral toxoplasmosis among 1,699 HIV-infected patients followed in the SEROCO and HEMOCO cohorts (1988-1995) was studied. It increased from 0.7 per 100 person-years in 1988 to 2.1 per 100 person-years in 1992, as a result of the increasing prevalence of patients with CD4 cell counts below 200/μL. It decreased thereafter to 0.2 per 100 person-years in 1995, while the proportion of patients receiving specific prophylaxis was increasing. A Toxoplasma antibody titer of >150 IU/mL was an important predictor of toxoplasmosis (adjusted relative risk [aRR], 3.6 [95% confidence interval, 2.1-6.0]), independent of a CD4 + cell count of 150 IU/mL, while the median CD4 cell count at onset of toxoplasmosis was 58/μL. Thus, disease was diagnosed 10 days to 74 months after the rise in Toxoplasma antibody titers. While the risk factors for development of toxoplasmosis remain incompletely defined, the importance of specific prophylaxis for patients with low CD4 cell counts and high Toxoplasma antibody titers is supported by these findings.

Molecular connectivity has been applied to the search of new compounds with activity against the protozoan Toxoplasma gondii, using a stepwise linear discriminant analysis (SLDA) which is able to classify a compound according its activity either as active or as inactive. Among the selected compounds, andrographolide and dibenzotiophene sulfone stand out, both with IC50 values lower than 1 microgram/ml, which are comparable to these of drugs such as sulfamethoxazole, pyrimethamine and trimethoprim, with IC50 values equal to 1.1, 0.04 and 2.31 micrograms/ml, respectively. These results confirm the usefulness of our topological approach for the selection and design of new-lead drugs active against Toxoplasma gondii.

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