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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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

3. Tauber MG, Sande MA. Pharmacodynamics of antibiotics in experimental bacterial meningitis--two sides to rapid bacterial killing in the cerebrospinal fluid. Scand J Infect Dis 1991; 74(suppl): 173-8. 4. Pfister HW, Fontana A, Tauber MG, Tomasz A, Scheld WM. Mechanisms of brain injury in bacterial meningitis: Workshop summary. Clin Infect Dis 1994; 19. 463-79. 5. Kim YS, Sheldon RA, Elliot BR, Liu Q, Ferriero IIM, Tauber MG. Brain damage in neonatal meningitis caused by group B meptococci in rats. J Neuropathol Exp Neurol 1995; 54: 531-9. 6. Nau R, Schmidt T, Tauber MG. Quinolone antibiotics in experimental pneumococcal meningitis in rabbits. Antirnicrob Agents Cheniother 1995; 3 9 593-7.

To determine the types of Enterocytozoon bieneusi strains associated with intestinal microsporidiosis, we developed a rapid and efficient approach for typing parasites obtained from stool specimens by PCR-restriction fragment length polymorphism (PCR-RFLP). Typing was based on DNA polymorphism of the ribosomal DNA internal transcribed spacer (ITS) region of E. bieneusi . RFLPs generated with two restriction enzymes ( Nla III and Fnu 4HI) in PCR-amplified ITS products were used to classify strains into different lineages. This approach was successfully used to differentiate 78 strains that had been obtained from the stools of 65 patients with intestinal microsporidiosis. Among the 78 strains, we found four genetically unrelated lineages, showing the genetic diversity of E. bieneusi . Type I strains of E. bieneusi were found in a majority of the samples, accounting for 51 (78%) of the 65 microsporidiosis cases. In contrast, type II, III, and IV strains were found in only 8 (12%), 3 (5%), and 3 (5%) cases, respectively. All strains of E. bieneusi we have tested so far fall into one of four different lineages, and this study shows that human intestinal microsporidiosis is most often associated with type I strains. PCR-RFLP analysis of the ITS region of E. bieneusi should be useful for epidemiological studies.

Objective Intestinal microsporidiosis due to Enterocytozoon bieneusi is a frequent cause of chronic diarrhoea in patients with HIV infection for which there is no available therapy. This study was designed to search for a drug with activity against this organism. Design Prospective open-labelled Phase II multicentre study. Setting University hospitals. Patients Sixty HIV-infected men with intestinal E. bieneusi infection. Interventions Ten drug regimens were consecutively tested orally for 3 weeks: albendazole plus metronidazole, sulphadiazine plus pyrimethamine, atovaquone, doxycycline plus nifuroxazide, itraconazole, flubendazole, chloroquine, paromomycin, sparfloxacin and fumagillin. Nine evaluable patients per regimen were required, but each patient could be enrolled up to three times in the study. Outcome measure Efficacy was assessed primarily by the clearance of E. bieneusi from stools and intestinal biopsies. The safety of each regimen was also assessed. Results Only purified fumagillin was able to clear E. bieneusi from stools as well as intestinal biopsies, whereas all other regimens failed to show antiparasitic efficacy. However, only four patients received fumagillin because of drug-induced thrombocytopenia. The four patients who received fumagillin remained free of E. bieneusi infection after a mean follow-up of 10 months. Conclusion Eradication of E. bieneusi from the intestinal tract of patients with HIV infection and persistent immunosuppression is an achievable goal. Our study allowed the identification of oral fumagillin as a potential treatment for intestinal microsporidiosis due to E. bieneusi.

Objective To study the usefulness of polymerase chain reaction (PCR) for the species identification of microsporidia in stool specimens obtained from HIV-infected patients with Enterocytozoon bieneusi or Encephalitozoon intestinalis infections. Setting Infectious disease clinic in a university hospital. Patients Thirty-seven stool specimens from 29 HIV-infected patients with microsporidiosis were tested. The diagnosis of microsporidian infection was made by light microscopy of stool specimens and species identification was made by transmission electron microscopy of duodenal biopsies. Sixty-one stool specimens from 45 HIV-infected patients without microsporidiosis served as controls. Methods PCR was performed using DNA extracted from stools with two primers sets, one specific for E. bieneusi and one specific for E. intestinalis. Results A 1265 base-pair fragment of the small subunit ribosomal RNA (rrs) gene could be amplified from all 31 stool specimens infected with E. bieneusi. In addition, a 930 base-pair fragment of the rrs gene could be amplified from all six stool specimens infected with E. intestinalis. The 61 control stools were negative with both primers. Conclusions These results suggest that a PCR based assay using species-specific primers sets can be used successfully for microsporidian species differentiation from stool specimens, thus obviating the need for invasive biopsy procedures.

The pathogenicity of 22 strains of Leishmania infantum from 11 HIV-infected and 11 immunocompetent patients with visceral (VL, n = 16) or cutaneous (CL, n = 6) leishmaniasis, belonging to 3 zymodemes (MON-1, n = 14; MON-29, n = 5; MON-33, n = 3), was studied using a murine model. For each strain 16-20 BALB/c mice were infected at day 0 (d0) by i.v. injection of 10(7) stationary-phase promastigotes. Parasite burdens were quantified in the spleen and liver of 4-5 mice of each strain at d7, d20, d60 and d90 or d100, using a sensitive culture microtitration technique. A great variability of infection profiles between strains was observed: (i) six strains showed a progressive infection, with a predominance of hepatic parasites at d7 or d20 (10(4)-10(6) g-1), then a continuous rise of splenic parasites reaching 10(5)-10(7) g-1 at d90 or d100 contrasting with a stagnation or decrease in the liver; (ii) ten strains gave a controlled infection with hepatic parasite burden reaching 10(4)-10(5) g-1 at d7 or d20, followed by a more or less rapid decline leading frequently to no detectable parasites; (iii) six strains resulted in other profiles, i.e., undetectable infection (n = 1) or low parasite loads (n = 4), or late occurrence of parasites in the spleen (n = 1). No relationship was observed between profile and growth characteristics in vitro or zymodeme of the strain. Strains originating from CL never gave a visceralizing pattern in mice, but belonged more frequently to the avirulent type compared to VL strains. Strains from HIV-infected patients were not less virulent than those from immunocompetent individuals. These results showed that the course of L. infantum infection varies markedly with intrinsic parasite factors that display striking intraspecific variability.

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.

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum . The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium -positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium -seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum , were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Aspergillus fumigatus spores in food may represent an infectious risk for neutropenic patients. We examined the efficiency of disinfection procedures applicable to foods for eradication of A fumigatus. Boiling and microwave treatment fully decontaminated an experimental spore suspension and naturally contaminated liquid foods (reconstituted dried food, herbal tea). Full decontamination of experimentally contaminated surfaces was only obtained with 70% ethanol or heating at 220°C for 15 minutes. Pepper was decontaminated when heated for 15 minutes at 220°C but not by microwaving. Fruit skin was partially decontaminated by 70% ethanol. We conclude that A fumigatus spores can be eradicated from food by heating to a temperature of at least 100°C. When foods cannot be exposed to high temperature or microwaving, ethanol only partially reduces the level of surface contamination.

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.

To the Editor: Diversity of genotypes among Pneumocystis jirovecii (human-specific Pneumocystis species) isolates mainly involves internal transcribed spacer (ITS) loci (1). Type Eg, one of the most frequently detected ITS genotypes, has been found worldwide (2). The locus of dihydropteroate synthase (DHPS) is also of interest because DHPS is the target of sulfonamides, the main drugs used to treat Pneumocystis pneumonia (PCP). Studies of the DHPS locus have found mutations at positions 165 and 171, which confer potentially lower sensitivity to sulfonamides to mutant P. jirovecii organisms (3). Airborne transmission of Pneumocystis ssp. has been demonstrated among animals and probably occurs among humans (4). Reports of clusters of PCP cases in hospitals (4,5) provide a rationale for considering the possibility of nosocomial P. jirovecii infections. Moreover, we recently quantified P. jirovecii in the air surrounding patients with PCP (6). Our findings suggested that the fungus is exhaled from infected patients and then spreads into their surrounding air. Because matches of P. jirovecii genotypes between pulmonary and air samples would strengthen these findings, we conducted DHPS and ITS typing of P. jirovecii isolates from PCP patients and from the air in their close environment. We assayed P. jirovecii DNA that we had previously detected in pulmonary samples (bronchoalveolar lavage and induced sputum) from 15 PCP patients and in 15 air samples collected 1 meter from each patient’s head (6). ITS genotyping was based on sequence analysis of ITS 1 and 2 regions after amplification with a nested PCR, cloning, and sequencing, as described (7). ITS alleles were identified by using the typing system by Lee et al. (2). DHPS genotyping was based on a PCR restriction fragment-length polymorphism assay that enables detection of mutations at positions 165 and 171, as described (8). Among the 15 pulmonary samples, ITS genotyping was successful for all 15; among these, 8 ITS genotypes were identified (Table). Type Eg was most frequently identified. Mixed infections, which correspond to detection of >1 genotype in a given sample, were detected in 5 samples. DHPS genotyping was successful for all 15 pulmonary samples. A wild genotype was identified in 9 samples, a 165 mutant genotype in 1 sample, and a 171 mutant genotype in 2 samples. Mixed infections were identified in the 3 remaining samples. Table Genotyping of Pneumocystis jirovecii in pairs of pulmonary and air samples from 15 patients with Pneumocystis pneumonia* Among the 15 room air samples, ITS genotyping was successful for 7; among these, 4 ITS genotypes were identified (Table). Type Eg was again most frequently identified. A mixed infection was detected in 1 of the 7 samples. These results enabled us to compare ITS genotypes for 7 pairs of pulmonary and air samples. A full match was found for 4 (57.1%) pairs of samples, and a partial match, defined as at least 1 common genotype for pulmonary and air samples in mixed infections, was found for 2 (28.6%) pairs. No matches were found for the remaining pair of samples. DHPS genotyping was successful for 6 of the 15 air samples. A wild genotype was identified in 4 samples, a 165 mutant genotype was identified in 1 sample, and a 171 mutant genotype was identified in 1 sample. These results enabled us to compare DHPS genotypes for 6 pairs of samples. A full match was found for these 6 pairs. DHPS and ITS genotype matches were found for 4 pairs. Several lines of evidence suggest that P. jirovecii is exhaled by infected patients and transmitted by the airborne route to susceptible persons (4). In the study reported here, ITS or DHPS genotype matches between pairs of pulmonary and air samples are consistent with the possibility that P. jirovecii organisms in the air originated from patients. DHPS mutants were detected in 6 (40%) of the 15 pulmonary samples; none of the15 patients had received sulfonamide treatment at the time of PCP diagnosis. These results were not unexpected because frequency of finding DHPS mutants in PCP patients in Paris who had no prior sulfonamide treatment is high (8). The exhalation of DHPS mutants from infected patients can spread potentially sulfonamide-resistant organisms. Matches of P. jirovecii genotypes in pairs of pulmonary and room air samples argue in favor of P. jirovecii exhalation by infected patients. The exhalation of P. jirovecii organisms emphasizes the risk for their nosocomial transmission. Our data provide additional arguments in favor of the application of measures to prevent the airborne transmission of P. jirovecii in hospitals.

Early evaluation of treatment efficacy in invasive aspergillosis (IA), a leading cause of morbidity and mortality in hematological patients, remains a challenge. We conducted a prospective study to evaluate the performance of different markers in predicting the outcome of patients with IA. Both clinical and biological criteria were assessed 7, 14, 21, and 45 days after inclusion in the study, and mortality was assessed at day 60. The association between baseline data and their evolution and the day 45 response to treatment was analyzed. A total of 57 patients (4 with proven, 44 with probable, and 9 with possible aspergillosis according to the revised EORTC/MSG [European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group] definitions) were included. At day 45, 30 patients (53%) were determined to be responders, 25 (44%) were nonresponders, and 2 were not able to be evaluated. Twenty patients died within the 60 days of follow-up. We found that a poor day 45 outcome was associated with patients who had high baseline serum galactomannan (GM) antigen levels and those receiving steroids at the time of IA. A consistently negative serum GM index was associated with a good outcome, and the day 14 clinical evaluation was predictive of the day 45 outcome. No association was found between Aspergillus antibodies or DNA detection and patients' outcome. We conclude that the GM index value at diagnosis of IA, GM index kinetics, and clinical evaluation at day 14 are good markers for predicting the outcome of patients with IA and should be taken into account for adapting antifungal treatment.

The identification of the causative organism in invasive pulmonary aspergillosis (IPA) is recommended. We investigated whether a mycologic diagnostic strategy could be optimized based on patient characteristics. Fifty-five patients were enrolled in a prospective study. The presence of Aspergillus in respiratory samples occurred more frequently in non-acute leukemia (AL) patients than in AL patients (P = .0003), and in patients with leukocyte counts more than 100/mm3 (P = .002). In a logistic regression model, these 2 factors appeared to be independent, with an adjusted odds ratio of 7.14 (95% confidence interval, 1.40-36.5) for non-AL patients and an adjusted odds ratio of 6.97 (95% confidence interval, 1.33-36.5) for patients with leukocyte counts more than 100/mm3. A positive mycologic result was also more frequent among patients with lung CT scan signs of airway-invasive disease than among other patients (P = .043). Airway-invasive signs were more frequent among non-AL patients (P = .049), whereas angioinvasive disease was more frequent among both AL patients (P = .01) and patients with leukocyte counts less than 100/mm3 (P = .001). A concomitant pulmonary infection was identified more frequently among non-AL patients (P = .005 vs allogeneic hematopoietic stem cell transplant and P = .048 vs others). Our results suggest that different strategies for diagnosing IPA should be considered based on the underlying condition.

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.

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.

Detection of Toxoplasma gondii in blood by means of the polymerase chain reaction (PCR) may facilitate the diagnosis and follow-up of cerebral toxoplasmosis in patients with AIDS. We evaluated this approach with seven patients with tissue culture-proven parasitemia, 14 patients with presumptive cerebral toxoplasmosis, and 17 healthy human immunodeficiency virus-positive controls. Each sample of blood was assayed on three different occasions by a PCR assay based on detection of the gene encoding the P30 surface protein. A positive PCR diagnosis required positivity in at least two of the three PCR tests. None of the controls had a positive PCR diagnosis, but six of the seven patients with parasitemia did. Cerebral toxoplasmosis was confirmed in 13 of the 14 patients with a presumptive diagnosis; diagnosis by PCR was positive before treatment for 9 of these 13 patients, whereas tissue culture was positive for only 1 patient. During treatment, blood samples were taken from 14 patients at regular intervals until day 12. PCR diagnosis became negative on ethidium-stained gels, but persistent signals were observed after hybridization, in some cases, for up to 12 days after initiation of therapy. PCR on venous blood could thus be a sensitive and noninvasive method for the diagnosis of cerebral and disseminated toxoplasmosis in AIDS patients and could be a potential tool for monitoring the effects of treatment.

Eighteen patients infected with human immunodeficiency virus and with chronic unexplained diarrhea were prospectively studied to investigate the prevalence and clinical and biologic features of intestinal microsporidiosis. All patients underwent extensive evaluation for bacterial, viral, and parasitic pathogens. Enterocytozoon bieneusi was found in 9 patients (50%; 95% confidence interval, 27-73) in stools and duodenal and jejunal biopsies. In 8 patients, it was the sole pathogen found. Other pathogens were also isolated from the intestinal tracts of 4 patients, but diarrhea remained unexplained in 6. Patients with intestinal microsporidiosis had significantly lower mean Karnofsky scores (69.4 vs. 85.5, P = .009), CD4 cell counts (18.6 vs. 209.8/microL, P = .02), and D-xylose absorption tests (0.13 vs. 0.36 g/L, P < .001) than did patients without intestinal microsporidiosis. Intestinal microsporidiosis appears to be a frequent cause of unexplained chronic diarrhea in patients with AIDS and is associated with diminished D-xylose absorption.