Pneumocystosis: a network survey in the Paris area 2003-2008.

The aims of this network group were to collect epidemiological data of PcP cases in 14 hospitals in the Paris area and to determine the Di-Hydro Pteroate Synthase (DHPS) genotypes, genetic markers for possible sulfamide resistance. From January 1, 2003 to December 31, 2008, 993 (mean 166/year) PcP cases have been reported. Sixty-five percent of patients were HIV-positive. The median count of CD4 lymphocytes was 32/mm(3) (30 in HIV-positive patients, 152 in HIV-negative patients). In HIV-positive patients, PcP revealed the HIV infection in 39%. Among 304 PcP occurring in HIV known infected patients, no prophylaxis was prescribed for 64%; cotrimoxazole prophylaxis had been prescribed to 47 patients but only one of them had the right compliance. In HIV-negative patients (264), corticosteroids were prescribed in 59% and cytotoxic chemotherapies in 34%; 78% did not receive prophylaxis. One hundred sixty nine tumoral pathologies and 116 transplantations were notified. The mortality rate was 16% at day 14 (13% in HIV-positive patients, 26% in HIV-negative patients). Mutations in DHPS genes were detected in 18.5% of samples; 12.5% of patients were infected with several strains. The total annual number of cases has been stable for five years but the proportion of HIV-negative patients increased from 25% to 43%.

Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients.

Diagnosis of pneumocystosis usually relies on microscopic demonstration of Pneumocystis jirovecii in respiratory samples. Conventional PCR can detect low levels of P. jirovecii DNA but cannot differentiate active pneumonia from colonization. In this study, we used a new real-time quantitative PCR (qPCR) assay to identify and discriminate these entities. One hundred and sixty-three bronchoalveolar lavage fluids and 115 induced sputa were prospectively obtained from 238 consecutive immunocompromised patients presenting signs of pneumonia. Each patient was classified as having a high or a low probability of P. jirovecii pneumonia according to clinical and radiological presentation. Samples were processed by microscopy and by a qPCR assay amplifying the P. jirovecii mitochondrial large-subunit rRNA gene; qPCR results were expressed as trophic form equivalents (TFEq)/mL by reference to a standard curve obtained from numbered suspensions of trophic forms. From 21 samples obtained from 16 patients with a high probability of P. jirovecii pneumonia, 21 were positive by qPCR whereas only 16 were positive by microscopy. Fungal load ranged from 134 to 1.73 × 10(6) TFEq/mL. Among 257 specimens sampled from 222 patients with a low probability of P. jirovecii pneumonia, 222 were negative by both techniques but 35 were positive by qPCR (0.1-1840 TFEq/mL), suggesting P. jirovecii colonization. Two cut-off values of 120 and 1900 TFEq/mL were proposed to discriminate active pneumonia from colonization, with a grey zone between them. In conclusion, this qPCR assay discriminates active pneumonia from colonization. This is particularly relevant for patient management, especially in non-human immunodeficiency virus (HIV)-infected immunocompromised patients, who often present low-burden P. jirovecii infections that are not diagnosed microscopically.

Fumagillin for treatment of intestinal microsporidiosis in renal transplant recipients.

We report 10 cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in renal transplant (RT) recipients who were treated with fumagillin. All patients presented with afebrile subacute diarrhea (median of 2 weeks), associated with abdominal cramps (n = 5), and weight loss (n = 6), a mean of 68 months after RT. The diagnosis was made by the identification of microsporidial spores in stools with the use of appropriate staining and confirmed by a specific polymerase chain reaction assay for E. bieneusi in 7 patients. Median CD4 cell count was 292 cells/mm(3). All patients received a median of 14 days of oral fumagillin (20 mg tid), and four patients also discontinued or tapered their immunosuppressive regimen (mycophenolate mofetil in 3, and azathioprine in 2). Clinical symptoms resolved rapidly with the clearance of microsporidial spores from stools in all patients. A severe but reversible thrombocytopenia was observed in one patient during fumagillin therapy, and another patient presented with abdominal cramps. Trough levels of tacrolimus measured in seven patients dropped below 5 ng/mL in six of them after 7-14 days of fumagillin. Intestinal microsporidiosis can cause subacute diarrhea in RT recipients. Fumagillin is an effective treatment with an acceptable safety profile, but monitoring of tacrolimus levels is warranted.

Microsporidiosis in solid organ transplant recipients: two Enterocytozoon bieneusi cases and review.

Microsporidiosis first came to prominence as an opportunistic infection in patients with acquired immunodeficiency syndrome. Microsporidia are now emerging pathogens responsible for severe diarrhea during solid organ transplantation. Two main clinical entities can be identified: infection by Enterocytozoon bieneusi, causing diarrhea with limited treatment options; and infection by Encephalitozoon intestinalis, which may disseminate and usually responds to albendazole treatment. We describe here 2 cases of microsporidiosis caused by E. bieneusi in a renal and a liver transplant recipient, respectively, in whom complete clinical efficacy of a short course of fumagillin therapy was obtained. Long-term microbiological eradication was assessed using classical methods and monitored using a real-time quantitative polymerase chain reaction-based method. Both patients experienced drug-induced thrombocytopenia, which resolved after withdrawal of the treatment. We also review the 18 other previously reported cases of microsporidiosis in transplant recipients. In case of persistent diarrhea in solid organ transplant patients, microsporidiosis should be considered. Based on the present experience, treating E. bieneusi infection with 7 days of fumagillin therapy is adequate to eradicate E. bieneusi in this context.

Assessment of cryptodiag for diagnosis of cryptosporidiosis and genotyping Cryptosporidium species.

The performance of a new commercial PCR-enzyme-linked immunosorbent assay (ELISA) (Cryptodiag; Bio Advance, France) for the diagnosis of cryptosporidiosis and the identification of Cryptosporidium hominis and C. parvum from stool samples was examined. This test is based on PCR amplification of Cryptosporidium DNA extracted from stools, followed by an ELISA based on hybridization with Cryptosporidium sp.-, C. hominis-, or C. parvum-specific probes. In spiking experiments, approximately five oocysts were detected either in water or in stool suspensions while assessing for the efficient removal of stool PCR inhibitors. No cross-reactivity was observed in the detection of C. parvum and C. hominis using the respective specific probes. Thirty-three fecal samples from patients with microscopically proven cryptosporidiosis and 118 from patients with or without other digestive protozoan infections were tested by Cryptodiag, blinded to the results of microscopy. Compared to microscopy, the sensitivity of Cryptodiag was 97.0% (32/33) and 100% (33/33), including the gray zone, and specificity was 98.3% (116/118) and 96.6% (114/118), including the gray zone. Among 34 positive results, Cryptodiag identified 19 due to C. hominis, 8 due to C. parvum, and 7 due to Cryptosporidium spp. Genotyping by Cryptodiag agreed with reference typing methods in 85% of cases of C. parvum or C. hominis infections. Cryptodiag proved to be reliable and sensitive for the diagnosis of cryptosporidiosis. The use of specific probes allowed the identification of C. hominis and C. parvum, i.e., the two main species responsible for human cryptosporidiosis, and rapidly provided information on the possible source of infection.

Isosporiasis in patients with HIV infection in the highly active antiretroviral therapy era in France.

BACKGROUND: Isosporiasis, a rare cause of diarrhoea among HIV-infected patients in the pre-highly active antiretroviral therapy (HAART) era, seems to be re-emerging. METHODS: A retrospective study was carried out for the period 1995-2003 in two hospitals in Paris to describe the prevalence, clinical characteristics and therapeutic outcome of isosporiasis in HIV-infected patients, and to compare the findings with those for cryptosporidiosis and microsporidiosis. RESULTS: The prevalence of isosporiasis increased from 0.4 per 1000 patients in the pre-HAART era (1995-1996) to 4.4 per 1000 patients in the HAART era (2001-2003), whereas the prevalence of cryptosporidiosis and microsporidiosis decreased. Compared with patients with either cryptosporidiosis (n=91) or microsporidiosis (n=58), patients with isosporiasis (n=28) more frequently originated from sub-Saharan Africa (72%), were more frequently female and heterosexual, and had a higher median CD4 count at diagnosis (142 cells/microL). All patients with isosporiasis presented with diarrhoea, which was severe enough to lead to hospital admission for 60% of them. Fever was uncommon (7%). All patients were treated for isosporiasis, 27 of them with cotrimoxazole. Relapse of isosporiasis occurred in six of 16 patients (38%) despite maintenance cotrimoxazole therapy and HAART. CONCLUSION: Isosporiasis in France occurs mostly in patients emigrating from sub-Saharan Africa and can induce severe diarrhoea. Relapse is common despite cotrimoxazole maintenance therapy.

Borreliosis: a rare and alternative diagnosis in travellers' febrile illness.

We report a case of borreliosis mimicking uncomplicated malaria in a patient returning from Mali. Identification of spirochetes through examination of a thick blood smear completed by an acridine-orange quantitative buffy coat allowed the diagnosis of borreliosis. All symptoms rapidly resolved following tetracycline therapy. Epidemiological and clinical features of borreliosis, diagnostic tools and management are discussed.

Late-onset eosinophilic chronic meningitis occurring 30 years after Taenia solium infestation in a white Caucasian woman.

Unlike solitary parenchymal cysts, chronic meningitis is unusual in patients with neurocysticercosis and may poorly respond to treatment. We report the case of neurocysticercosis characterized by severe headache and chronic eosinophilic meningitis occurring 30 years after infestation with Taenia solium. The patient showed considerable improvement following treatment with albendazole and prednisone.

Occurrence of Pneumocystis jiroveci pneumonia after allogeneic stem cell transplantation: a 6-year retrospective study.

Pneumocystis jiroveci pneumonia (PCP) has become a rare opportunistic infection due to the efficacy of prophylactic regimens. We conducted a 6-year retrospective study at our institution. A total of 13 cases of PCP were diagnosed among 519 patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) (2.5%). In three patients, PCP occurred within the first 5 months following HSCT. These severely immunocompromised patients were receiving prophylaxis and had concomitant aspergillosis that caused rapid death in two of them. In 10 other patients, PCP occurred a median of 14.5 months after HSCT. In all these patients, PCP prophylaxis had been discontinued, mainly because of the suspected bone-marrow toxicity of the prophylactic regimen. Median CD4+ T cell count was 131/microl at diagnosis. Seven of these 10 patients were receiving immunosuppressive therapy for chronic graft versus host disease and three had a relapse of their hematological malignancy. One patient died from PCP despite high doses of cotrimoxazole. We conclude that PCP is still occurring after allogeneic HSCT, mainly as a late complication in patients in whom PCP prophylaxis had been prematurely discontinued. Long-term PCP prophylaxis should be maintained in patients receiving immunosuppressive drugs, and in those with low CD4+ T cell counts or a relapse of their hematological malignancy.

Detection of Microsporidia, cryptosporidia and Giardia in swimming pools: a one-year prospective study.

In order to estimate the rate of microsporidia, cryptosporidia and giardia contamination of swimming pools, sequential samples of water were collected during a one-year period in six different swimming pools in Paris, France. Fourty-eight samples were submitted to filtrations. Eluates were examined for microsporidia using polymerase chain reaction (PCR) and for cryptosporidia and giardia using immunofluorescence staining. One of 48 specimens was positive for microsporidia. Using DNA sequence analysis, unknown microsporidia species were identified, which were close to an insect microsporidia Endoreticulatus schubergi. One sample was positive for cryptosporidia and none were positive for giardia. This study shows a low level of swimming pool water contamination by microsporidia, cryptosporidia or giardia, demonstrating the efficacy of cleaning filtration and disinfection procedures used in French swimming pools.

Search for Enterocytozoon bieneusi infection in wild monkeys in Cameroon.

Faecal samples collected from 42 wild monkeys in Cameroon were examined for microsporidia by light microscopy (using Weber trichrome and Uvitex 2B stains) and by PCR (using Enterocytozoon bieneusi specific primers). None of the 42 samples was positive, suggesting that wild monkeys do not represent a major reservoir for microsporidia in Central Africa.

Toxoplasmic pneumonitis leading to fatal acute respiratory distress syndrome after engraftment in three bone marrow transplant recipients.

Toxoplasmosis is a rare but severe complication of bone marrow transplantation. Here, we report three patients in whom toxoplasmic pneumonitis developed, leading to fatal acute respiratory distress syndrome (ARDS). All patients had positive pretransplantation tests for Toxoplasma gondii and were therefore at risk to develop toxoplasmosis reactivation. They all recovered from aplasia, but soon after they died from brutal and severe ARDS. The possible role of an immunopathologic response to T gondii in the lungs in triggering ARDS is discussed.Early screening of parasitemia using highly sensitive polymerase chain reaction methods in seropositive patients with unexplained fever may be needed.

Molecular characterization of Cryptosporidium isolates obtained from humans in France.

Cryptosporidium parvum is usually considered the agent of human cryptosporidiosis. However, only in the last few years, molecular biology-based methods have allowed the identification of Cryptosporidium species and genotypes, and only a few data are available from France. In the present work, we collected samples of whole feces from 57 patients from France (11 immunocompetent patients, 35 human immunodeficiency virus [HIV]-infected patients, 11 immunocompromised but non-HIV-infected patients) in whom Cryptosporidium oocysts were recognized by clinical laboratories. A fragment of the Cryptosporidium 18S rRNA gene encompassing the hypervariable region was amplified by PCR and sequenced. The results revealed that the majority of the patients were infected with cattle (29 of 57) or human (18 of 57) genotypes of Cryptosporidium parvum. However, a number of immunocompromised patients were infected with C. meleagridis (3 of 57), C. felis (6 of 57), or a new genotype of C. muris (1 of 57). This is the first report of the last three species of Cryptosporidium in humans in France. These results indicate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.

Evidence of different Enterocytozoon bieneusi genotypes in patients with and without human immunodeficiency virus infection.

We classified 100 Enterocytozoon bieneusi isolates into five genotypes by a PCR-restriction fragment length polymorphism method. Type I strains were encountered only in human immunodeficiency virus (HIV)-infected patients, whereas type II strains were more frequently found in non-HIV-infected patients (75 versus 10%, respectively; P < 10(-4)), suggesting differences in the epidemiology of E. bieneusi among these patients.

[Microsporidiosis]. / Les microsporidies.

OPPORTUNISTIC PARASITES: Microsporidia are primitive eukaryotic parasites widespread in a large range of animal species. These opportunistic parasites can cause infections in humans, mainly in immunocompromised patients. PATHOGENIC SPECIES: Four microsporidian species are important in human pathology, Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi and Encephalitozoon hellem. LABORATORY DIAGNOSIS: A difficult task, laboratory diagnosis is based on direct microscope visualization of the parasite. Special stains not used in routine practice are required for identifying spores. TREATMENT: Species differentiation, achieved with the polymerase chain reaction technique, is necessary to select the appropriate treatment. Treatment of the most common microsporidiosis (caused by Enterocytozoon bieneusi) with fumagillin is currently under assessment in an ANRS clinical trial.

Detection of microsporidia in surface water: a one-year follow-up study.

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.

Trial of oral fumagillin for the treatment of intestinal microsporidiosis in patients with HIV infection. ANRS 054 Study Group. Agence Nationale de Recherche sur le SIDA.

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 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.

Genetic homology among thirteen Encephalitozoon intestinalis isolates obtained from human immunodeficiency virus-infected patients with intestinal microsporidiosis.

The ribosomal DNA internal transcribed spacer sequences of 13 unrelated Encephalitozoon intestinalis isolates obtained from human immunodeficiency virus (HIV)-infected patients with intestinal microsporidiosis were analyzed by gene amplification and DNA sequencing. Among these isolates, we found only one genetic lineage which suggests that E. intestinalis may have a clonal distribution in HIV-infected patients.