The effect of essential oils from Thymus broussonetii Boiss on transmission of Toxoplasma gondii cysts in mice.

In this study, we have evaluated the effect of essential oils of Thymus broussonetii Boiss, an endemic plant of Morocco in experimental transmission of Toxoplasma gondii cysts in mice. These oils were obtained by hydrodistillation and were administered to mice at 20 microg/animal orally at the time of infection and for several days thereafter. This resulted in total absence of intracerebral cysts in mice who received the essential oils signifying that these essential oils of thyme have a blocking effect on the appearance of the cysts. In addition, no abnormality was observed in the control mice who received the essential oils of thyme.

A technique for dating toxoplasmosis in pregnancy and comparison with the Vidas anti-toxoplasma IgG avidity test.

A comparative evaluation of 384 selected sera was performed using the Beckman Coulter Access and Abbott Axsym Toxo-IgG assays. The Axsym assay yields positive early results following infection, while the Access assay gives higher titres during chronic infection. The ratio between the two complementary tests, Axsym Toxo-IgG/Access Toxo-IgG (Ax/Ac), was compared with the Vidas anti-Toxoplasma IgG avidity index (AI). The Ax/Ac ratio decreased progressively as the time between infection and sampling increased. The mean Ax/Ac values (+/-SE) were 2.50 (+/-0.26), 2.14 (+/-0.13), 2.33 (+/-0.22), 1.34 (+/-0.09), 1.32 (+/-0.10), 0.92 (+/-0.08) and 0.74 (+/-0.07) for groups of sera sampled at 1, 2, 3, 4-5, 6-8, 9-12 and 13-24 months, respectively, after infection in pregnant women. These values were much smaller for cases with chronic infection (>24 months), i.e., 0.56 (+/-0.03), 0.44 (+/-0.04) and 0.53 (+/-0.04), respectively, for pregnant women and immunodepressed patients with and without reactivation. Taking a ratio of 1 as a threshold for recent infection, the patients in the groups sampled at 1, 2 and 3 months had Ax/Ac ratios >1 in 49/50 (98%), 53/55 (96.4%) and 36/36 (100%) cases, respectively. Thus, an Ax/Ac ratio of <1 in serum from a pregnant woman allows a recent infection (<3 months) to be excluded. This technique has the advantage of yielding positive results that develop much more rapidly than the AI, thereby helping to reassure large numbers of pregnant women and avoiding costly and unnecessary prophylactic treatment and follow-up.

[Dry sausage mould hypersensitivity pneumonitis: three cases]. / Pneumopathie à la fleur de saucisson: trois observations.

INTRODUCTION: Hypersensitivity pneumonitis (HP) can occur as a consequence of inhaling a wide range of different antigens. The clinical diagnosis is based on five main criteria as proposed by the GERM'O'P. CASE REPORTS: We present in this report three cases of occupational hypersenstitivity pneumonitis caused by inhalation of dry sausage mould. Lung infiltrates were observed in each case on CT scanning, with a lymphocytic or mixed inflammatory picture in bronchoalveolar lavage fluid, and specific antibodies (precipitins) against extract from dry sausage mould (which notably contains Penicillium). In all three cases, the condition improved after reducing exposure to the allergen and, in two cases, after the administration of corticosteroid therapy. CONCLUSION: Exposure to dry sausage mould occurs in work places where salami is made and should be considered as a possible cause of hypersenstitivity pneumonitis.

Efficacy of environmental measures to decrease the risk of hospital-acquired aspergillosis in patients hospitalised in haematology wards.

This study evaluated a multidisciplinary strategy to decrease the rate of invasive pulmonary aspergillosis (IPA) among adult patients hospitalised in two haematology wards in a single 560-bed building at the University Hospital of Saint-Etienne. Upgrading of the air filtration system and construction of an air-lock chamber at the entrance to the unit were completed during 1994. In 1995, specific hygienic measures were introduced during hospital building work, including the use of plastic barriers, watering during demolition work, reduction of pedestrian traffic in construction areas, and the wearing of high-efficiency filtration masks by immunosuppressed patients when outside the protected unit. This strategy was evaluated by a prospective survey of IPA cases between 1993 and 2001, coupled with environmental surveillance. The number and risk-level of hospital renovation projects increased between 1995 and 2001 (p < 0.01). In parallel, the rate of IPA decreased globally in the haematology unit from 0.85% (1.19/1,000 patients) in 1993 to 0.28% (0.21/1,000 patients) in 2001. The incidence of IPA decreased significantly between 1993-1996 and 1997-2001 (p 0.02, Mann-Whitney test). These results show that a multidisciplinary approach involving engineers, infection control practitioners, mycologists and clinicians enables IPA rates among patients hospitalised in haematology wards to be significantly decreased.

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

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

Efficiency of blood culture bottles for the fungal sterility testing of corneal organ culture media.

BACKGROUND/AIM: The consequences of fungal contamination of an organ cultured cornea, though exceptional, are often disastrous for the recipient. Consequently, eye banks often quarantine corneas for 10 days or more before passing them for grafting. This period, though detrimental to the endothelial cell density of the delivered cornea, is necessary to detect contamination using conventional microbiological methods. The authors previously validated the use of a pair of aerobic and anaerobic blood bottles for sensitive and rapid detection of bacteria. To allow a short quarantine period, it remained only to optimise detection of fungi. The authors aimed to compare sensitivity and rapidity of fungal contamination detection by three methods: blood bottles, Sabouraud, and daily visual inspection of the organ culture medium. METHODS: Four inocula (10(6), 10(4), 10(2), 10 colony forming unit (CFU) per ml) of 11 fungi (Candida albicans, C tropicalis, C glabrata, Saccharomyces cerevisiae, Rhodotorula rubra, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus niger, A fumigatus, A flavus, Acremonium falciforme) were inoculated in a commercial organ culture medium containing a coloured pH indicator (CorneaMax, Eurobio, Les Ulis, France). The real live fungal inoculum was verified immediately after inoculation. After 48 hours at 31 degrees C, samples of the contaminated media were inoculated in three blood bottles: Bactec Aerobic/F, Bactec Mycosis IC/F, and Bactec Myco/F Lytic (Becton Dickinson, Le Pont de Claix, France), then placed in a Bactec 9240 rocking automat, and in four Sabouraud media (solid and liquid, 28 degrees C and 37 degrees C) with daily observation. Contaminated organ culture media were also checked daily for any change in turbidity and/or colour. Experiments were performed in triplicate. RESULTS: Mycosis IC/F and Myco/F Lytic bottles were neither faster nor more sensitive than the aerobic bottle. The three methods were positive for all inocula, even the lowest (viable inoculum below 10 CFU/ml for each fungus). Contamination was detected within 24 hours by the aerobic bottles in 91% (40/44), by Sabouraud in 98% (43/44) (no significant difference) and by visual inspection in 66% of cases (29/44) (p<0.001 with the two others). Maximum times to detection were 46, 48 and 72 hours respectively. CONCLUSION: This study further counters the preconception that fungal contamination is hard to detect in corneal organ culture media. This study is the last step in validating the use of a pair of blood bottles for the sterility testing of organ culture media, this time for fungi. Their use should make it possible to shorten microbiological quarantine and thus deliver corneas with higher endothelial cell density, without increasing the risk of recipient contamination.

Seroprevalence of T. gondii in sheep from Marrakech, Morocco.

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite transmitted by food. Concerning this parasite, there are few studies done in Morocco. In this study, 261 sera from sheep intended for consumption in Marrakech were subjected to the Toxoplasma ELISA based serology test for the detection of anti-T. gondii specific IgG confirming a past infection. Of the total tested 72 (27.6%) sera were positive for IgG. This result shows that the seroprevalence approaches the world average and is similar to what is found in other cities of Morocco. This has prompted us to investigate other animal species in the region in order to evaluate the degree of contamination by this parasite as well as the potential risk incurred on consumption of their meat.

Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75, 48, 30, 24 and 22 kDa).

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

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

Detection of circulating antigens of Toxoplasma gondii in human infection.

Seventy-nine serum specimens from pregnant women and 29 from immunocompromised patients (12 from graft recipients and 17 from patients with acquired immunodeficiency syndrome) were classified into three groups according to their serologic status to Toxoplasma gondii as determined by immunofluorescence and an enzyme-linked immunosorbent assay (ELISA): no antibodies (group 1), acute acquired infection (group 2), and reactivation (group 3). These samples were tested for the presence of circulating antigens (CAg) of T. gondii by capture ELISA and immunoblotting. The presence of CAg was detected by at least one of the two techniques in six of 31 subjects in group 1, 51 of 68 subjects in group 2, and seven of nine subjects in group 3. Of a total of 108 serum specimens, 28 were found to be T. gondii-positive by capture ELISA, 57 by immunoblotting, and 21 by both techniques. Among the nine polypeptides detected by immunoblotting, 38 recognized p14, 17 recognized p8, and 16 recognized p8, and 16 recognized p30. These results demonstrate that the detection of CAg can aid in the diagnosis of infection by T. gondii in humans, especially in immunocompromised patients whose serologic response can be impaired.

Comparison of PCR, capture ELISA and immunoblotting for detection of Toxoplasma gondii in infected mice.

PCR was compared with capture ELISA and immunoblotting for the detection of Toxoplasma gondii in sera of acutely infected mice. One hundred animals were inoculated intraperitoneally with 5000 trophozoites of RH strain and five of them were killed every 3 h from 3 h to 21 h post infection (p.i.), and every day from day 1 to day 7 p.i.. No assay detected the parasite from 3 h p.i. to 15 h p.i. PCR was the most sensitive assay and detected the T. gondii from 18 h p.i., whereas the other assays detected it only from day 1.

Experimental model of congenital toxoplasmosis in guinea-pigs: use of quantitative and qualitative PCR for the study of maternofetal transmission.

Maternofetal transmission of Toxoplasma gondii was assessed in pregnant guinea-pigs, with a gestational period of 65 +/- 5 days. A total of 56 female guinea pigs was infected by the intraperitoneal route (RH strain), by the oral or the intraperitoneal route (Prugniaud strain; PRU) or by the oral route (76K strain). Inoculation was performed 90 +/- 18 days or 30 +/- 9 days before the onset of gestation or 20 +/- 6 days or 40 +/- 6 days after. Gestational age was determined by a progesterone assay. Parasite loads (fetal brain and liver) were assessed by nested PCR and real-time PCR quantification on Light Cycler was performed with a SYBR Green I technique. The 76K strain appeared to be the most virulent in the model: the neonatal survival rate was 31%, in contrast to 53% and 68% for the PRU and RH strains, respectively. The percentage of survival of neonates for all strains taken together was lower after inoculation at 40 days' gestation (25%) than at 20 days' gestation (77%). Whatever the strain, maternofetal transmission determination was greater with nested PCR (54% for RH, 84% for PRU and 86% for 76K strains) than with real-time quantitative PCR (31% for RH, 66% for PRU and 76% for 76K strains). However, real-time quantitative PCR showed that neonatal parasite load was greater with the cystogenic strains (76K, PRU) and that high hepatic load (> 10000 parasites/g) was often associated with disease severity (11 of 12 cases). Therefore, this technique may provide an important element in understanding this congenital disease.

Influence of the quantity of nonspecific DNA and repeated freezing and thawing of samples on the quantification of DNA by the Light Cycler.

Quantification of DNA in real-time using the Light Cycler is increasingly being used for the detection and follow-up of various infectious and other diseases. We evaluated the effect of two parameters, namely the presence of nonspecific DNA and prior repeated freezing and thawing on the accurate quantification of DNA extracts from the RH strain of Toxoplasma gondii by the SYBR Green I and the Hybridization Probe techniques. For both parameters, a high copy number sample containing 5x10(5) parasites/extract and a low copy number sample containing 100 parasites/extract were tested. Reliable quantification was possible in the presence of up to 200 ng of nonspecific DNA by the SYBR Green I technique and up to 1000 ng by the Hybridization Probe technique as compared to the company threshold of 50 and 500 ng, respectively. As tissue samples usually contain more than 200 ng of nonspecific DNA, the ideal choice is the Hybridization Probe technique. The stability of DNA extracts after repeated freeze-thaw cycles was found to be dependent on the volume in which they were stored. Samples stored in 100-microl total volumes were not stable after 3 freeze-thaw cycles, whereas those stored in 1-ml total volumes were stable after 14 freeze-thaw cycles.

Identification and initial characterization of Pneumocystis carinii soluble antigens in rabbit serum and lung lavage.

Lung lavage and serum samples from Azathioprin-treated (acute-phase infection) and untreated (non acute-phase infection) rabbits were used in the immunoblotting technique to look for Pneumocystis carinii (Pc) soluble antigens, using rabbit polyclonal antibodies raised against rabbit-derived Pc antigens and labeled with peroxidase. Analysis of the supernatant of lavage fluid after centrifugation to sediment intact organisms revealed components of approximately 80, 60-65, 55, 39 and 27 kDa in acute-phase samples. The components in the regions of 80, 60-65, 55 kDa and to a lesser extent 39 kDa were also present in non acute-phase lung lavage samples. In acute-phase serum samples, a major component of 80 kDa and minor components of about 65 and 39 kDa are detectable. The 80 and 65 kDa components are also detectable in some of the serum samples from the untreated rabbits. Immunofluorescent staining with FITC-conjugated affinity-purified antibodies to the 80, 60-65, 55, 39 or 27 kDa-components showed that they shared epitopes with both Pc cysts and trophozoites. The affinity-purified antibodies also cross-reacted in immunoblotting with several antigens in the Pc whole preparations. The putative Pc soluble antigens in serum and lung lavage were then isolated by affinity chromatography with polyclonal antibodies to Pc. Preliminary characterization of the column-extracted antigens revealed complete inactivation by trypsin whereas only the 55 and 80 kDa antigens bind to Concanavalin A. In conclusion, the results of this study suggest that Pc soluble antigens are present even in non acute-phase samples and only the low-molecular weight antigens (39 and 27 kDa) seem specific for the acute-phase. These findings are consistent with previous investigations reported by others that development of Pc could occur in nonimmu-nosuppressed rabbits.

Kinetic of circulating antigens by capture ELISA and immunoblotting in murine toxoplasmosis.

Capture ELISA and immunoblotting techniques were applied for the detection of Toxoplasma gondii (T.g) circulating antigens (CAG) in serum specimens of OF-1 mice infected with virulent RH, or cystogenic PGD strains. The capture ELISA assay allowed to detect CAG from the first day of infection to the death of animals with the two T.g strains, although CAG rates were higher when the RH strain was used. Immunoblotting confirmed these findings. Moreover, immunoblotting allowed to identify 7 CAG (MW: 22 kDa to 110 kDa) in serum specimens of mice infected with the RH strain and only 3 (MW: 75 kDa to 110 kDa) in serum specimens of mice infected with the PGD strain. These results are discussed in the light of the evolutive ways of experimental infection by T.g.