Jamal Hafid

Acquired resistance to voriconazole and itraconazole in a patient with pulmonary aspergilloma

We describe the development of resistance in an Aspergillus fumigatus strain, originally sensitive to itraconazole and voriconazole, recovered from a case of pulmonary aspergilloma treated with voriconazole. A G448S mutation on the cyp51A gene was detected by sequencing. Frequent culture and in vitro antifungal susceptibility testing is suggested for early detection of the development of multi-azole resistance in patients on long-term therapy for A. fumigatus infections.

Reliability of Immunoglobulin G Antitoxoplasma Avidity Test and Effects of Treatment on Avidity Indexes of Infants and Pregnant Women

ABSTRACT The immunoglobulin G antitoxoplasma avidity test (Vidas; BioMérieux) is an immunoenzymatic test useful for excluding acute infection after the onset of pregnancy. The avidity index (AI) is the ratio of the signal in a test sample washed with urea, which disrupts low-avidity complexes, to that washed without urea. An AI of >0.3 is taken to mean that infection had occurred more than 4 months ago. The increase of the AI with time and the influence of the different treatments given to pregnant women and their newborns were evaluated. A total of 59 pregnant women (271 sera) and their 60 neonates (199 sera) were tested from 1998 to 2002. There were five groups of women based on the type and duration of treatment given. Thirteen pregnant women (group 1) did not receive any treatment, 15 (group 2), 11 (group 3), and 17 (group 4) women received treatment with spiramycin (9 MIU/day) for 0.5 to 2, 2.5 to 5, and 5.5 to 8 months, respectively, and the last 3 women (group 5) received tritherapy (pyrimethamine-sulfonamide and spiramycin alternatively) for 1.5 to 2.5 months. All of the maternal sera collected in the first 6 months had an AI of <0.30, with a mean of 0.07 (range, 0.01 to 0.21). The increase was slow (0.02/month), and there was no significant difference when comparisons were made between the treatment groups. Neonates with proven maternofetal transmission had an increasing AI, unlike those without transmission. However, long-term therapy with pyrimethamine-sulfonamide, as opposed to treatment with spiramycin alone, was found to slow down the progression of the AI. An AI of >0.2 is sufficient to exclude acute infection in pregnant women. In neonates, it is not of major use to diagnose congenital infection; however, it could be a good indicator of compliance and efficacy of treatment of infected infants.

Behavioral, histopathological and biochemical impairments observed in mice envenomed by the scorpion: Hottentota gentili (Pallary, 1924).

Hottentota gentili is a black scorpion which has been considered as dangerous specie by many authors. However there are no data regarding minimal lethal dose and effects of the scorpion venom till now. We therefore aimed, by the present investigation, to assess on the one hand, the LD50 of H. gentili venom by sublethal injection and the effects on some vital organs, by a histological and a biochemical tools. On the other hand, the possible neurobehavioral impairments, in Swiss mice, 3 h, 6 h and 12 h following envenomation. The LD50 of H. gentili scorpion venom was found to be 0.46 mg/kg by subcutaneous injection route. Venom produced focal fragmentation of myocardial fibers, while lungs showed rupture of the alveolar structure. Intestines showed selective histopathological changes. Concomitantly, there was a significant rise in the serum enzymes levels, as well as hyperkalemia and a high level of plasma albumine and creatine. Proteinuria was also observed. The observed behavioral effects were a hypoactivity in the both experiments 30 min and 3 h after injection. The envenomation produced an increased immobility time only 30 min and 3 h post injection in the tail suspension test (TST).

Toxoplasma gondii antigens: Analysis by SDS-PAGE and immunoblotting; Relation with circulating antigens

Cytoplasmic antigenic extracts (CAE) and membranar antigenic extracts (MAE) from Toxoplasma gondii (T.g) purified trophozoites RH strain, were obtained after hypotonic lysis and sonication. Analysis in Polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie blue staining revealed 21 polypeptides (PP) in CAE and 8 in MAE, whereas the immunoblotting technique detected 28 PP in CAE and 23 in MAE. Immunoblotting is then more sensitive than Coomassie blue staining. This finding is particularly marked for MAE and could be explained by the high antigenicity of some PP that have a lower concentration than the threshold sensitivity of SDS-PAGE. In contrast, some PP in sufficient amount to be detected by SDS-PAGE did not show any antigenic activity since not found by immunoblotting. The molecular weight (MW) ranged from 8 to 130 kDa for cytoplasmic PP and from 4 to 110 kDa for membranar PP. Circulating antigens (CAG) from serum specimens of mice infected with the same strain of T.g were analysed by immunoblotting in comparison with CAE and MAE profiles. From the 7 bands obtained with CAG, 4 were shown to correspond to CAE PP, 2 to common CAE and MAE PP and only 1 to MAE PP (MW: 30 kDa).

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.

Evaluation of a New Immunochromatography Technology Test (LDBio Diagnostics) To Detect Toxoplasma IgG and IgM: Comparison with the Routine Architect Technique

ABSTRACT A study comparing the ICT (immunochromatography technology) Toxoplasma IgG and IgM rapid diagnostic test (LDBio Diagnostics, France) with a fully automated system, Architect, was performed on samples from university hospitals of Marseille and Saint-Etienne. A total of 767 prospective sera and 235 selected sera were collected. The panels were selected to test various IgG and IgM parameters. The reference technique, Toxoplasma IgGII Western blot analysis (LDBio Diagnostics), was used to confirm the IgG results, and commercial kits Platelia Toxo IgM (Bio-Rad) and Toxo-ISAgA (bioMérieux) were used in Saint-Etienne and Marseille, respectively, as the IgM reference techniques. Sensitivity and specificity of the ICT and the Architect IgG assays were compared using a prospective panel. Sensitivity was 100% for the ICT test and 92.1% for Architect (cutoff at 1.6 IU/ml). The low-IgG-titer serum results confirmed that ICT sensitivity was superior to that of Architect. Specificity was 98.7% (ICT) and 99.8% (Architect IgG). The ICT test is also useful for detecting IgM without IgG and is both sensitive (100%) and specific (100%), as it can distinguish nonspecific IgM from specific Toxoplasma IgM. In comparison, IgM sensitivity and specificity on Architect are 96.1% and 99.6%, respectively (cutoff at 0.5 arbitrary units [AU]/ml). To conclude, this new test overcomes the limitations of automated screening techniques, which are not sensitive enough for IgG and lack specificity for IgM (rare IgM false-positive cases).

Analysis of Pneumocystis carinii antigens by CIEP, SDS-PAGE and immunoblotting

Pneumocystis carinii (Pc) from rabbit and rat lungs were analyzed by counterimmunoelectro-phoresis (CIEP), Polyacrylamide gel electrophoresis and immunoblotting. In CIEP, considerable cross-reactivity was demonstrated using rabbit polyclonal antisera raised against the rabbit and the rat-derived Pc components. In immunoblotting, both the rat and the rabbit Pc antigens also showed reactivity with the two antisera. Rabbit-derived Pc antigens of about 24-27,39,42-45, 55, 110 and 116 kDa were revealed with infection-derived rabbit sera. The 39, 42-45 and 116 kDa antigens were further recognized by both human and rat immune sera. In addition, many human sera detected the rabbit Pc antigen of 42-45 kDa. Using rat infection-derived sera, rat Pc antigens of 39, 55 to 60, 110 and 116 kDa were identified. The sera from Pc infected rabbits and humans recognized the bands in the regions of 39, 55 and 116 kDa. Carbohydrate residues were further analyzed by Concanavalin A (Con A)-binding experiments, periodate Schiff (PAS) and Alcian Blue (AB) stainings. The 55 and 116 kDa bands of both rabbit and rat Pc strongly reacted with Con A and were stained by PAS indicating the presence of hydroxyl and mannosyl/glycosyl residues. The AB staining-bands were 39, 42-45 and 55 kDa for rabbit Pc isolates and 39, 45, 55-60 and 70 kDa for rat Pc isolates, indicating the presence of carbohydrate residues with acidic function. The rabbit Pc immunodominant 39, 55 and 116 kDa antigens then appeared to be glycoproteins with characteristics similar to those of their counterparts in rats. The implications of these findings are discussed.

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.