Hospital-acquired Aspergillus fumigatus infection: can molecular typing methods identify an environmental source?

Aspergillus fumigatus infection in hospitalized immunocompromised patients often raises suspicion regarding the potential for hospital acquisition. Hospital staff have an important responsibility in implementing preventive measures, especially since the advent of current legislation concerning hospital-acquired infections. There have been high expectations that molecular typing methods might determine the source of Aspergillus fumigatus, a ubiquitous mould. The aim of the present epidemiological study, was therefore, to identify the origin(s) of Aspergillus infection in six well-documented patients. All the clinical strains (N=33), and those from hospital (N=14) and home environments (N=34) were isolated according to a standardized protocol and typed by sequence-specific DNA primer analysis. The results confirmed the huge biodiversity of the A. fumigatus population, and consequently the difficulty in ascertaining a hospital source of the infection, as opposed to infections due to other Aspergillus species less frequently encountered.

A longitudinal study of lung transplant recipients infected with Aspergillus: genetic polymorphism of A fumigatus.

BACKGROUND: Aspergillus infection is a well-known complication of lung transplantation and remains associated with high mortality rates. Molecular typing methods are required to elucidate the complex epidemiology of Aspergillus disease in lung transplant recipients. METHODS: Eight lung transplant recipients from one hospital were followed for A fumigatus colonization or infection. Forty-four sequential isolates from these patients were selected and typed by three molecular methods (random amplified polymorphic DNA, sequence-specific DNA primer and multi-locus enzyme electrophoresis). RESULTS: Sixteen different types were identified of which 14 were specific to 1 patient. A factorial correspondence analysis showed that variability between sequential isolates from a single patient was as high as between isolates from the other patients. Lung transplant recipients presented many different genotypes, reflecting the environmental diversity of A fumigatus. Nevertheless, throughout their follow-up, 2 of the 8 lung transplant recipients harbored a common genotype that was not replaced by others. CONCLUSIONS: These results confirm the important genetic polymorphism of the A fumigatus population. The observed genotypes were not related to the type of Aspergillus disease or anti-fungal treatment used nor to the outcome of the patient. These data confirm that all A fumigatus molecular types present the same pathogenic risk.

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.

Detection of RAPD markers correlated with chloroquine resistance in Plasmodium falciparum.

We described the use of the random amplified polymorphic DNA (RAPD) technique on Plasmodium falciparum DNA to detect genetic markers for chloroquine-resistant strains. Fourteen RAPD primers were tested, three of which generated banding patterns correlated with chloroquine resistance. To measure this correlation, the RAPD profiles were analyzed using the Nei and Li similarity coefficient. Detection of distinctive RAPD bands allowed us to synthesize specific PCR primers to be used on whole-blood samples. Two primer sets were synthesized and tested on sensitive and resistant strains for their ability to amplify the DNA fragment corresponding to the RAPD marker. These results suggest that RAPD and PCR techniques can be used as powerful tools for the detection of genetic markers associated with drug resistance.

Apoptosis related to chloroquine sensitivity of the human malaria parasite Plasmodium falciparum.

As chemoresistance of Plasmodium falciparum to chloroquine has arisen, new ways of combating the infection are needed. Similarities exist between the multidrug resistance of mammalian cells and chloroquine resistance of P. falciparum, based on the occurrence of internucleosomal deoxyribonucleic acid (DNA) breakdown and the ability of some anticancer drugs and chloroquine to induce apoptosis. Using chloroquine, oligonucleosomal DNA fragmentation was observed with a sensitive strain of P. falciparum, but not with a resistant one. This suggests that apoptosis may be involved in the action of chloroquine on the parasite.

Identification of differentially transcribed RNA and DNA helicase-related genes of Plasmodium falciparum.

Chloroquine antimalarial action was assessed by the analysis of changes in gene expression. With this aim, Plasmodium falciparum cultures were submitted to chloroquine and to other stresses to determine which transcripts were specifically induced. P. falciparum in vitro control culture was compared to cultures where chloroquine was added and to cultures where serum was omitted, or where higher partial oxygen pressure was used, and, finally, at a temperature of 40 degrees C instead of 37 degrees C. Poly (A)+RNAs were reverse-transcribed and detected by the differential display technique. Two specific cDNAs were obtained and cloned, and a part of the genes was sequenced. The deduced protein, referred to as Pfhel-1, was related to a RNA helicase and was thought to be involved in protein translation control. The second deduced protein, called Pfhel-2, possessed consensus sequences of ATP-dependent helicase domains. Pfhel-2 may be involved either in mitotic control or in DNA repair. The possible roles of both helicase-related genes in chloroquine therapeutic activity are discussed.

Evidence for expression of a Ras-like and a stage specific GTP binding homologous protein by Plasmodium falciparum.

Plasmodium falciparum, the parasite responsible for the most severe form of malaria, undergoes an asexual multiplication in man and a sexual one in mosquito. The asexual cycle can be reproduced in vitro. The present work reports the isolation of a small guanosine triphosphate-binding protein in Plasmodium falciparum extracts. This protein, a 21,000 M(r) Ras-like molecule, was revealed by western blotting in each stage of the intraerythrocytic asexual life cycle. Conversely, a 46,000 M(r) G alpha subunit of a heterotrimeric GTP-binding protein was found to be expressed during a short period from mature schizonts to free merozoites. In order to provide additional evidence for the presence of these GTP-binding proteins in Plasmodium falciparum cultures and also to determine the kinetics, we tested two toxins that are involved in the cellular signalling transduction. We observed that pertussis toxin increases P. falciparum growth, whereas cholera toxin induces crisis forms, and subsequent parasite death within the following 24 h.

Estrone sulfatase activity in normal and abnormal endometrium.

We investigated the activity of estrone sulfatase in normal and pathological endometrium. In normal endometrium, the estrone sulfatase activity [pmol E1 X min-1 X (mg prot)-1] was 23.13 +/- 8.44 (mean +/- SD). An increase (p less than 0.01) of estrone sulfatase activity (62.81 +/- 21.97) was noted in mild endometrial hyperplasia. In focal hyperplasia (when the measurements were performed in the normal endometrial biopsies) such an increase was not noted (19.10 +/- 5.33). Estrone sulfatase activities of moderate hyperplasia (25.30 +/- 11.40) and endometrial neoplasia (30.30 +/- 9.57) were in the same range as in normal endometrium. Treatment with progestagen simultaneously reduced hyperplasia and estrone sulfatase activity. But when morphologically abnormal endometrium persisted after treatment, estrone sulfatase activity remained increased. The increase of estrone sulfatase activity appeared to be specific to mild endometrial hyperplasia. The role of estrone sulfatase in the pathogenesis of endometrial hyperplasia is discussed.

Estrone sulfate concentrations in plasma of normal individuals, postmenopausal women with breast cancer, and men with cirrhosis.

Estrone sulfate is quantitatively the most important estrogen in plasma. A method for its determination in human plasma is described, and the precision, accuracy, sensitivity, and specificity are defined. Free steroids were extracted from plasma with diethyl ether and steroid sulfates were isolated with use of Vlitos' reagent (methylene blue in dilute H2SO4/Na2SO4 solution). After enzymic hydrolysis, estrone was isolated by chromatography on Celite and measured by radioimmunoassay. The mean concentrations (nmol/L +/- 1 SD) of estrone sulfate were 2.51 +/- 0.90 for plasma from 13 women in follicular phase, 5.33 +/- 1.55 for 17 women in luteal phase, 0.89 +/- 0.60 for 44 postmenopausal women, and 0.96 +/- 0.43 for 24 postmenopausal women with breast cancer. Results for postmenopausal women with or without breast cancer did not differ significantly. For 13 normal men, estrone sulfate concentrations were 2.62 +/- 0.79 nmol/L, and for a group of 19 cirrhotic men the mean value was 1.43 +/- 0.95 nmol/L, significantly lower than normal.

[Antenatal administration of betamethasone. Effects upon neonatal blood glucose in premature infants (author's transl)]. / Administration anténatale de bétaméthasone. Effet sur la glycémie néonatale des prématurés.

This effect was studied in 23 preterm neonates at the age of 2--8 hours. Betamethasone was administered at a dose of 12 mg intramuscular once or several times before delivery. This glycemia was compared with the glycemia of 52 control preterm infants studied at the same period. In the control preterm neonates, the mean (+/- 1 S.D.) blood glucose level was 0.51 +/- 0.26 g/l versus 0.53 +/- 0.24 g/l in the preterm infants pretreated with betamethasone. The incidence of glycemia less than 0.30 or 0.20 g/l was not significantly different in the two groups. These data show that prenatal betamethasone therapy has no harmful or diserable effect on the neonatal glycemia of preterm infants.