Human parvovirus B19 infections are common in the general population, and infection during pregnancy may cause hydrops fetalis and fetal death. To initiate adequate treatment, accurate laboratory diagnosis is essential. The most sensitive tests are nested PCR systems, but these assays provide semiquantitative results at best. A parvovirus B19 DNA assay was developed based on the real time TaqMan PCR. This method was calibrated on the basis of serial plasmid dilutions and tested with an international parvovirus B19 standard. The assay was capable of quantifying parvovirus B19 DNA from one to about 5 x 10(7) genome equivalents per reaction (corresponding to 100 to 5 x 10(9) genome equivalents per ml serum). Samples from 51 pregnant women with suspected acute parvovirus B19 infection were tested, and positive PCR results were obtained in at least one of the materials investigated in 41 cases. The median viral DNA load in maternal blood samples was 1.3 x 10(4) copies/ml (range 7.2 x 10(2)-2.6 x 10(7)). Maternal virus DNA concentration was not associated with the presence of maternal symptoms and/or fetal complications. As the stage of infection was not known in the majority of cases, our data do not exclude an association between peak levels of parvovirus B19 DNA and the development of complications. Maternal sera and corresponding fetal material were available for concurrent testing from 15 DNA-positive cases: in most fetal samples, viral DNA concentrations were several orders of magnitude higher (up to 2.1 x 10(12) copies/ml) compared to the corresponding maternal blood samples.
We recovered two isolates (EP1 and EP2) of Escherichia coli from the same patient that had identical pulsed-field gel electrophoresis patterns but required different MICs of ciprofloxacin (CIP): 16 and 256 mg/liter for EP1 and EP2, respectively. Both isolates had mutations in the quinolone resistance-determining regions of GyrA (Ser83Leu and Asp87Tyr) and ParC (Ser80Ile), but not in those regions of GyrB or ParE. Isolate EP2 was also more resistant to chloramphenicol, tetracyclines, cefuroxime, and organic solvents. A deletion of adenine (A) 1821 was found in marR of isolate EP2, which resulted in an 18-amino-acid C-terminal deletion in the MarR protein. The causative relationship between ⌬A1821 and the Mar phenotype was demonstrated both by the replacement of the wild-type marR by marR ⌬A1821 in isolate EP1 and by complementation with the wild-type marR in trans in isolate EP2. In isolate EP2 complemented with wild-type marR, susceptibility to chloramphenicol was restored completely, whereas susceptibility to CIP was restored only incompletely. Northern blotting demonstrated increased expression of marA and acrAB but not of soxS in isolate EP2 compared to EP1. In conclusion, the deletion of A1821 in marR in the clinical isolate EP2 caused an increase in the MICs of CIP and unrelated antibiotics. Presumably, the C-terminal part of MarR is necessary for proper repressor function.
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