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.

Association between obesity and vulnerability and serologic response to influenza vaccination in older adults.

BACKGROUND: Serologic response to influenza vaccination declines with age. Few other host factors are known to be associated with serologic response. Our objective was to determine whether obesity and vulnerability independently predicted serologic response to influenza vaccination. METHODS: Adults ≥ 50 years were recruited during the 2008-2009 influenza season. Subjects provided pre- and post-vaccination sera for measuring antibody titers to 2008-2009 vaccine components. Body mass index (BMI) was calculated as weight (kg)/height (m(2)). Data were collected on vulnerability using the vulnerable elders survey (VES13). Logistic regression evaluated the associations between obesity and vulnerability and the serologic response to vaccination (both seroprotection and seroconversion), adjusting for gender, age, comorbidities, pre-vaccination titer, and site. RESULTS: Mean (± standard deviation) age of 415 study subjects was 65 ± 10 years; 40% were obese. Mean BMI was 29 ± 5.6 kg/m(2); mean VES13 was 1.6 ± 1.8. The proportions of subjects who seroconverted and had seroprotective titers were 40% and 49%, respectively, for A/Brisbane/59 (H1N1); 73% and 80% for A/Brisbane/10 (H3N2); and 34% and 94% for B/Florida. Modified VES-13 (score 0-10, with 10 being most vulnerable) was not associated with seroprotection against H1N1 or H3N2, and VES-13 was directly associated with seroconversion to H1N1 but not H3N2 or B. Obesity (BMI ≥ 30 kg/m(2) vs. BMI 18.5-30 kg/m(2)) was not associated with seroprotection for H1N1 or H3N2; obesity was directly associated with seroconversion to H3N2 but not H1N1 or B. Age was inversely associated with seroprotection and seroconversion against H1N1 and with seroconversion to influenza B. CONCLUSION: Based on this sample of older healthy subjects, there were no consistent relationships between VES 13 or obesity and either seroprotection or seroconversion to three influenza vaccine antigens.

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.

Active transport of cimetidine and ranitidine into the milk of Sprague Dawley rats.

Diffusion determines the extent of accumulation into milk for most xenobiotics. However, cimetidine (CM) and ranitidine (RN) have been reported to accumulate to an extent greater than expected in rat and human milk, suggesting an active transport mechanism. In the present study, lactating Sprague Dawley rats were used in a random crossover design to characterize CM and RN active transport. Rat milk-to-serum ratios (M/S) (29.3 +/- 3.2 vs. 13.0 +/- 6.0; P < .05) and systemic clearance, Cls (12.9 +/- 1.2 vs. 4.6 +/- 1.0 ml/min, P < .05), were significantly reduced when exposed to a higher steady state infusion regimen of CM (0.4 and 30 mg/hr, respectively). By contrast, a infusion regimen of RN (0.4 and 30 mg/hr, respectively) produced modest, but not statistically significant, reductions in M/S (12.7 +/- 3.8 vs. 9.0 +/- 2.6; P > .05) and Cls (12.2 +/- 1.5 vs. 9.9 +/- 2.7 ml/min; P > .05). In a third set of rats, CM M/S (30.4 +/- 2.7 vs. 27.5 +/- 4.6; P > .05) and Cls (12.5 +/- 2.8 vs. 10.7 +/- 4.8 ml/min; P > .05), were marginally reduced by a concomitant RN infusion regimen (30 mg/hr) when compared with CM steady state infusions alone (0.4 mg/hr). By contrast, RN M/S (16.1 +/- 2.0 vs. 10.5 +/- 2.0; P < .05) and Cls (11.0 +/- 1.3 vs. 7.1 +/- 0.9 ml/min, P < .05), were significantly reduced by a concomitant CM infusion regimen (30 mg/hr) when compared with RN steady state infusions alone (0.4 mg/hr). Models for M/S and Cls as a function of CM steady state serum concentration were proposed and fitted to the data. Values for the maximum transport velocity of the transport system (Tmax') and the apparent dissociation constant (Km) for the M/S relationship were 326 and 55 micrograms/ml, respectively. For the Cls relationship, estimates of the nonsaturable clearance component (Clns), the maximum velocity of the saturable elimination process (Vmax), and Km were 3.6 ml/min, 135 micrograms/min, and 16 micrograms/ml, respectively. These observations provide evidence that CM and RN milk transfer can be saturated and inhibited, which would be consistent with the hypothesis that these compounds are actively transported across mammary epithelial cells into rat milk.

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.

The genetic relationships between Echinostoma caproni, E. paraensei, and E. trivolvis as determined by electrophoresis.

Adults of Echinostoma caproni, E. paraensei, and E. trivolvis were processed for starchgel electrophoresis. Ten enzyme systems representing 12 structural loci were examined using three different buffer systems. E. paraensei and E. caproni were found to be genetically inbred as indicated by the lack of heterozygosity in individual worms. All three taxa showed fixed differences indicating they are distinct species. Fixed differences were found between E. paraensei and E. caproni in six enzyme systems, between E. paraensei and E. trivolvis in five enzyme systems, and between E. trivolvis and E. caproni in five enzyme systems. Phenic relationships among the three species showed E. caproni was genetically more similar to E. trivolvis than to E. paraensei.