Multicenter Evaluation of the Novel ETEST Fosfomycin for Antimicrobial Susceptibility Testing of Enterobacterales, Enterococcus faecalis, and Staphylococcus Species.

Fosfomycin is a phosphonic acid derivative active against a wide spectrum of Gram-positive and Gram-negative pathogens. It is used for the treatment of uncomplicated urinary tract infections (uUTI) or severe infections by oral or intravenous (i.v.) administration. In order to improve its performance and robustness, the fosfomycin strip, an antibiotic gradient diffusion strip, was redeveloped and evaluated in the multicenter study summarized in this paper. ETEST fosfomycin (ETEST FO) clinical performance was evaluated by three study sites on 152 Enterococcus faecalis, 100 Staphylococcus spp. and 330 Enterobacterales in comparison with the CLSI and EUCAST agar dilution reference method. Referring to FDA performance criteria, the ETEST FO achieved 91.0% of essential (EA) and 99.0% of categorical agreement (CA) for Escherichia coli. In addition, 98.0% EA and 93.4% CA were achieved for E. faecalis, with no very major errors (VME) or major errors (ME). According to EUCAST breakpoints for intravenous fosfomycin use, Enterobacterales and Staphylococcus spp. also met ISO acceptance criteria for EA and CA (EA 91.5%, 94.0%, respectively, and CA 98.0% for both). A VME rate of 8.8% was observed for Enterobacterales but the MICs were within EA. A trend to predict lower MICs for Citrobacter spp., E. coli and Salmonella enterica and to predict higher MICs for Klebsiella pneumoniae MICs was observed, while ETEST FO should not be used for Enterobacter cloacae, because of low EA and a high VME rate. The study results support the efficiency of the novel ETEST FO, making it an easy-to-handle tool as a substitute to the classical agar dilution method.

Comparison of a real-time PCR method with serology and blood smear analysis for diagnosis of human anaplasmosis: importance of infection time course for optimal test utilization.

Anaplasmosis and ehrlichiosis are emerging tick-borne diseases with clinically similar presentations caused by closely related pathogens. Currently, laboratories rely predominantly on blood smear analysis (for the detection of intracellular morulae) and on serologic tests, both of which have recognized limitations, for diagnostic purposes. We compared the performance of a published real-time PCR assay that incorporates melt curve analysis to differentiate Anaplasma and Ehrlichia species with blood smear and serologic methods in an upper Midwest population. Overall, 38.5% of the specimens selected for evaluation had one or more tests that were positive for anaplasmosis. The PCR positivity for all specimens was maximal (21.2%; 29/137) during the early acute phase of illness (0 to 4 days since illness onset) and significantly less frequent (11.5%; 20/174) during later phases (>4 days since illness onset). All positive specimens were Anaplasma phagocytophilum; no Ehrlichia species were identified. The real-time PCR detected 100% of infections that were detected by blood smear analysis (14/14) and broadened the detection window from a maximum of 14 days for smear positivity to 30 days for PCR. Additional infections were detected by real-time PCR in 12.9% (11/85) of smear-negative patients. There was poor agreement between the real-time PCR assay and serologic test results: 19.8% (19/96) and 13.7% (29/212) of seropositive and -negative patients, respectively, were PCR positive. Seropositivity increased with increasing days of illness, demonstrating that serologic detection methods are best utilized during presumed convalescence. Our results indicate that the optimal performance and utilization of laboratory tests for the diagnosis of anaplasmosis require knowledge regarding time of symptom onset or days of illness.

Linkage map organization of expressed sequence tags and sequence tagged sites in the mosquito, Aedes aegypti.

A composite genetic linkage map for the yellow fever mosquito Aedes aegypti was constructed based on restriction fragment length polymorphism (RFLP), single nucleotide polymorphism (SNP) and single strand conformation polymorphism (SSCP) markers. The map consists of 146 marker loci distributed across 205 cM, and includes several morphological mutant marker loci. Most of the genetic markers are derived from random cDNAs or Ae. aegypti genes of known function. A number of markers are derived from random genomic DNAs, including several cloned RAPD-PCR fragments, and also several cDNAs from Drosophila melanogaster. Most of the random cDNAs (80.2%) have high BlastX sequence identities to known genes, with the majority of matches to genes from D. melanogaster. Access to sequence data for all markers will facilitate their continued development for use in high-throughput SNP marker analyses and also provides additional physical anchor points for an anticipated genome sequencing effort.

A comparison of the adult and miracidial stages of Echinostoma paraensei and E. caproni.

Adult Echinostoma paraensei and Echinostoma caproni were grown in outbred mice and golden hamsters to compare size, growth rates, infectivity, and habitat selection. Antagonistic responses between the 2 species were investigated by concurrent infections in mice. Miracidial stages were compared for developmental stages, hatching responses, and behaviour to light and gravity. Size differences and growth rates were significantly different in both mice and hamsters. Mice proved to be better hosts for E. caproni and hamsters for E. paraensei. In mature infections, E. paraensei adults localized in the duodenum and E. caproni in the ileum of both mice and hamsters. In concurrent infections of mice, E. paraensei adults were significantly smaller than in single species infections beyond 14 days post-infection, while E. caproni adults were either equal to or larger than those in single species infections. On the other hand, E. paraensei were recovered in larger numbers in concurrent infections than in single species infections, while the reverse was found for infectivity of E. caproni adults. Miracidia of E. paraensei developed at the same rate as those of E. caproni in both light and dark cultures, but E. paraensei hatched much sooner when exposed to light. No miracidia hatched from cultures kept in the dark, indicating light is needed to stimulate the hatching process. All light-stimulated cultures exhibited a circadian hatching pattern from 1100 to 1600 hours. Cultures maintained in the dark past 11 days did not hatch when exposed to light. Miracidia of E. paraensei showed a positive phototaxis but no response to gravity. This comparison of life cycle stages leads us to conclude that E. paraensei and E. caproni are distinct species.