First sequence-confirmed case of infection with the new influenza A(H1N1) strain in Germany.

Here, we report on the first sequence-confirmed case of infection with the new influenza A(H1N1) virus in Germany. Two direct contacts of the patient were laboratory-confirmed as cases and demonstrate a chain of direct human-to-human transmission.

Detection of herpesvirus DNA in cerebrospinal fluid and correlation with clinical symptoms.

BACKGROUND: Novel PCR techniques can detect minute quantities of herpesvirus DNA in cerebrospinal fluid (CSF). The clinical significance of such findings is not always clear. PATIENTS AND METHODS: (a) Investigation of clinical characteristics of 76 patients with herpesvirus DNA detection in CSF. (b) Screening for herpesvirus DNA in CSF samples of 208 patients without clinical signs of herpesvirus infection. RESULTS: (a) Eleven of 76 herpesvirus-DNA-positive patients did not show symptoms usually associated with the detected virus (HSV-1/2, n = 5; EBV, n = 6). (b) Two of 208 patients without hint for herpesvirus infection had HHV-6 DNA of low concentration in CSF. CONCLUSIONS: The detection of low-level herpesvirus replication in CSF by highly sensitive PCR assays requires critical evaluation.

[Polymerase chain reaction (PCR) for microbiological diagnosis in refractory infectious keratitis: a clinical study in 16 patients]. / Die Polymerase-Kettenreaktion (PCR) zur mikrobiologischen Diagnostik einer persistierenden infektiösen Keratitis: Eine klinische Studie bei 16 Patienten.

BACKGROUND: The identification of the causative pathogen in infectious keratitis is possible in only 60% of the cases. The aim of this study was to show if this number increases by the use of PCR. PATIENTS AND METHODS: In a series of 16 eyes with infectious keratitis corneal specimens were collected for culture and PCR. Serology (HSV, VZV, and Borrelia) was performed in all eyes, with exception of the 4 eyes presenting an acute form of keratitis, which obviously was bacterial origin. RESULTS: In all 4 cases of acute keratitis the causative pathogen (Pseudomonas aeruginosa) was detected by both culture and PCR. Of the remaining 12 eyes PCR was capable to identify the causative pathogen in 11 eyes. In 3 eyes herpes simplex virus was detected, in 3 eyes Moraxella catharalis, in 2 eyes Borrelia burgdorferii, in 2 eyes varizella zoster virus, and in 1 eye Bartonella henselae. Culture was positive in only 2 eyes, infected by Moraxella catharalis. CONCLUSIONS: PCR is a useful supplement in the microbiological diagnostic of infectious keratitis, in particular if only a small amount of pathogens are available (non-acute form) or if the eye has been treated by antibiotics prior to the microbiological diagnostic.

In vivo increase in resistance to ciprofloxacin in Escherichia coli associated with deletion of the C-terminal part of MarR.

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 DeltaA1821 and the Mar phenotype was demonstrated both by the replacement of the wild-type marR by marR DeltaA1821 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.

A method of assessing the efficacy of hand sanitizers: use of real soil encountered in the food service industry.

In many outbreaks of foodborne illness, the food worker has been implicated as the source of the infection. To decrease the likelihood of cross-contamination, food workers must clean and disinfect their hands frequently. To ensure their effectiveness, hand disinfectants should be tested using rigorous conditions that mimic normal use. Currently, several different methods are used to assess the efficacy of hand disinfectants. However, most of these methods were designed with the health care worker in mind and do not model the specific contamination situations encountered by the food worker. To fill this void, we developed a model that uses soil from fresh meat and a means of quantifying bacteria that is encountered and transferred during food preparation activities. Results of studies using various doses of para-chloro-meta-xylenol and triclosan confirm that the method is reproducible and predictable in measuring the efficacy of sanitizers. Consistent, dose-dependent results were obtained with relatively few subjects. Other studies showed that washing hands with a mild soap and water for 20 s was more effective than applying a 70% alcohol hand sanitizer.

Prevalence of precore mutants in anti-HBe-positive hepatitis B virus carriers in Germany.

Hepatitis B virus (HBV) precore mutants are associated often with highly productive infection in hepatitis B surface antigen (HBsAg) carriers lacking hepatitis B e antigen (HBeAg) but positive for anti-HBe, rendering serological identification of infectious individuals unreliable. Although considered initially to be limited mostly to the Mediterranean area, more recent studies suggest a significant presence of these mutants in northern European countries. The sequence of the precore region was determined and examined for mutations from HBV isolates of 99 German chronic HBsAg carriers positive for HBV-DNA and either HBeAg (n = 15) or anti-HBe (n = 84). In addition, clinical data of individuals carrying wild-type virus and those with precore mutants were compared. HBV precore mutants were found in more than half (44/84) of all HBeAg-negative, anti-HBe-positive virus carriers. There was no difference between carriers of wild-type and precore mutant HBV in the level of viremia or in the clinical course of chronic infection. In conclusion, HBV precore mutants are common in Germany and can therefore present a diagnostic problem for serological testing. However, precore mutants do not appear to have a detrimental effect on the course of chronic HBV infection.

[Surgical treatment of epistaxis]. / Operacyjne leczenie krwawien z nosa.

The authors present 17 patients with nose bleeding who had to be treated surgically, because conservative procedures turned out unsuccessful. Causes of bleeding, types of conservative procedures, as well as types of operations are described. Surgical treatment was fully efficacious in 12 patients (70%).

[Treatment of recurrent nose bleeding caused by Rendu-Osler-Weber's disease in patients treated at the ENT department of the Regional Hospital in Opole]. / Postepowanie lecznicze w przypadku nawrotowych krwawien z nosa w przebiegu choroby Rendu-Oslera-Webera na podstawie badan przeprowadzonych na Oddziale Laryngologii Szpitala Wojewódzkiego w Opolu.

The authors describe a method of surgical treatment of nosebleeds caused by Rendu-Osler-Weber's disease. Free skin graft of the thigh region was used for closure of nasal septal defects. A new dermatoplasty technique allows to cover both sides of the nasal septum with a single skin graft. The diseased septal mucosa is denuded with a scalpel, but the underlying perichondrium is preserved. Excellent exposure of the nasal septum is obtained via a modified external rhinoplasty approach. This new dermatoplasty technique described by dr Bridger is a modification of traditional Saunders dermatoplasty. In three patients treated by this method at our department the transplantation ended successful.

Quantitative PCR : a survey of the present technology.

The polymerase chain reaction (PCR) is a powerful tool for the amplification of trace amounts of nucleic acids, and has rapidly become an essential analytical tool for virtually all aspects of biological research in experimental biology and medicine. Because the application of this technique provides unprecedented sensitivity, it has facilitated the development of a variety of nucleic acid-based systems for diagnostic purposes, such as the detection of viral (1) or bacterial pathogens (2), as well as genetic disorders (3), cancer (4), and forensic analysis (5). These recently developed systems open up the possibility of performing reliable diagnosis even before any symptoms of the disease appear, thus considerably improving the chances of success with treatment. For many routine applications, particularly in the diagnosis of viral infections, the required answer is the presence or the absence of a given sequence in a given sample. Therefore, PCR is in able for the early diagnosis of HCV infection (6), HSV encephalitis (7), or HIV infection of babies of HIV-positive mothers (8). On the other hand, since even minute amounts of DNA are detected, the medical interpretation of positive results for widespread infectious agents like CMV (9) or HHV6 (10) turned out to be rather difficult.

Competitive PCR quantitation utilizing a microtiter plate based format for the detection of PCR products.

In microbiology, the polymerase chain reaction (PCR) has become an important tool for the analysis of clinical samples. For example, it led to the detection of Hepatitis-B Virus DNA (HBV-DNA) in patients with serological patterns not previously associated with active infection (1). PCR-based assays are also more sensitive than conventional tests for the detection of many other infective agents, like Hepatitis-C Virus (HCV), herpes viruses, or Legionella pneumophila (2). In some cases, however, the detection of the nucleic-acid sequences of a certain infectious agent is not very informative. Here is a quantitative information of the amount of nucleic acid needed. DNA quantitation is useful for monitoring the viral load during antiviral therapy (3), in estimating the degree of infectiousity of individuals (4,5), and (e.g., for herpes viruses) to differentiate between latent and active infection. Owing to its sensitivity, quantitative PCR is the most sensitive technique for the quantitation of nucleic acids and therefore has been investigated in detail.

Delayed facial palsy following uneventful middle ear surgery: a herpes simplex virus type 1 reactivation?

In rare cases, a facial palsy appears a few days after uneventful middle ear surgery. The reason for this delayed palsy is unclear. One hypothesis is that it results from a reactivation of herpes simplex virus type 1 (HSV-1) in the geniculate ganglion of the facial nerve. From 1987 to 1996, in the course of over 1,800 middle ear operations, we observed 7 ipsilateral delayed facial palsies and investigated 5 of them using immunologic and virologic methods, including the polymerase chain reaction (PCR). We could detect HSV-1 genome with the nested primer PCR in the tongue swabs of 4 of the 5 examined patients with delayed facial palsy. The immunologic changes in these palsies are also compatible with a reactivation of HSV-1. We conclude that minimal stimulation of the facial nerve during middle ear surgery could result in a reactivation of HSV-1 in the geniculate ganglion, which may in turn lead to a facial palsy.

[Laryngeal chondrosarcoma]. / Chrzestniakomiesak krtani.

The authors showed the laryngeal chondrosarcoma in own materials.

Quantitative PCR. A survey of the present technology.

PCR-based amplification of nucleic acids has had a major impact in almost every field of basic research and has already found extensive applications in the area of clinical diagnosis. For many of these applications, quantitative data are sought to relate the quantity of amplified product to the amount of original target nucleic acid present in the sample. Since the PCR methodology with its exponential nature can be adapted for this purpose, a lot of different strategies have emerged in the last few years for sensitive and specific PCR product detection and quantification. Basic strategies, including the use of external and internal standards, are presented with respect to statistical aspects, and the advantages as well as the limitations of individual protocols are discussed. Furthermore the suitability of conventional laboratory techniques, such as gel systems or HPLC, nonradioactive labeling procedures, and the principles of advanced solid-phase-mediated strategies for the precise determination of amplification products, are outlined with the help of selected examples.

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Basic structure.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 and of DSM 20068 was isolated by phenol/water extraction and hydrophobic-interaction chromatography. The preparations from both strains have an identical structure: a 1,3-linked poly(glycerophosphate) chain phosphodiester-linked to Glc-(alpha 1-2)Glc(alpha 1-3)acyl2Gro as the lipid anchor. The chain is substituted with D-alanine ester and glycosyl residues which comprise mono-, di-, tri- and tetra-alpha-D-glucopyranosyl residues with (1-6) interglycosidic linkages. The glycosylglycerols were released with 48% (by mass) hydrofluoric acid, separated and characterized by a combination of chemical procedures and modern techniques of 1H-NMR and 13C-NMR spectroscopy. The alpha-isomalto-oligosaccharides add a novel motif to lipoteichoic-acid chain substituents. 1H-NMR and 13C-NMR spectroscopy also provided a detailed picture of the basic glycosylated poly(1,3-glycerophosphate) diglucosylglycerol. It proved a single unbranched chain structure, provided evidence for the chain length, the extent of glycosylation, the structure of the lipid anchor and the site of attachment of the poly(glycerophosphate) chain on the lipid anchor. Owing to its unique glycosyl substituents the lipoteichoic acid may serve as a taxonomic marker for the redefined species S. sanguis (formerly S. sanguis type I).

Isomalto-oligosaccharide-containing lipoteichoic acid of Streptococcus sanguis. Microheterogeneity and distribution of chain substituents.

The lipoteichoic acid of Streptococcus sanguis DSM 20567 contains a poly(glycerophosphate) chain, with 49% of the glycerophosphate residues being substituted with D-alanine ester, 35% with alpha-D-glucopyranosyl and alpha-isomalto-oligosaccharide residues. Analysis of molecular species by affinity chromatography on concanavalin A showed all chains to be substituted and alanine ester and glycosyl residues to be present on the same rather than on separate chains. Molecular species varied in the length of the poly(glycerophosphate) chain, the extent of glycosylation, and had a constant alanine-ester content. An alkali-hydrolysis procedure revealed a distribution pattern between random and regular for the glycosyl substituents and suggested a similar distribution for the alanyl residues which occupy the free positions between the glycosyl substituents.

Effect of calcium citrate-malate on skeletal development in young, growing rats.

It has been previously demonstrated that calcium from calcium citrate-malate (CCM), a mixture of calcium, citric acid and malic acid, is better-absorbed than calcium from calcium carbonate (CaCO3) in humans and in rats. It was of interest to determine if this differential in absorption would result in differences in bone development under chronic feeding conditions. The present study was designed to compare CCM with CaCO3 for effects on bone development in weanling female C/D rats fed either CCM or CaCO3 at 0.3 or 0.6% dietary Ca for 4 or 12 wk. There was a nonsignificant trend for rats fed CCM to weigh more and have larger bones than rats fed CaCO3. Histologic evaluation of cortical and trabecular bone revealed normal bone formation in all rats. Trabecular bone was significantly affected by calcium level and source. The 0.3% Ca diets (either source) resulted in reduced trabecular bone volumes in tibias. After 4 wk, rats fed CCM had 23-25% more trabecular bone than rats fed CaCO3. By 12 wk, the difference was even greater; rats fed CCM had 44-47% more trabecular bone than rats fed CaCO3. Dietary calcium source did not affect cortical bone. It is concluded that because of its positive effects on bone, CCM is a more bioavailable calcium source than CaCO3.

Decreased antibody formation in iron-deficient rat pups--effect of iron repletion.

Rats were fat diets containing 6, 12, or 250 ppm iron throughout gestation and lactation. On day 17, pups immunized with sRBC were used to determine antibody synthesis by the Jerne plaque assay. In both iron-deficient groups, antibody formation was decreased by at least 50% compared to controls. For 3 weeks beginning on day 21, iron-deficient pups were fed either a control diet (35 ppm iron) or the same iron-deficient diet as fed to the dam. IgG and IgM formation was only slightly improved in repleted rats and remained significantly below that of rats fed the control diet throughout the experiment. In contrast, 250 ppm iron pups fed an iron-deficient diet postweaning had significantly decreased IgG and IgM production compared to littermates fed a control diet postweaning. Maternal iron deficiency during the critical pre- and postnatal growth periods may result in long-term impairment of humoral immunity that is not corrected by dietary iron repletion after weaning.

Cellular growth in iron-deficient rats: effect of pre- and postweaning iron repletion.

Effects of iron deficiency and repletion pre- and postweaning on cell growth in young rats were studied. Pregnant dams were fed 6 or 250 ppm iron. On d 2 of lactation, half of the dams in each group were fed the opposite diet. On d 17, cell growth in the crossed-over groups was similar to controls showing that cellular development is impaired only when the iron deficiency is present during gestation and lactation. In a second experiment pup littermates of dams fed 6 (D), 12 (M) and 250 (C) ppm iron were weaned to either the same diet as fed to their dams DD, MM or CC; repleted with iron DC, MC; or fed the deficient diet CD until 42 d of age. After dietary iron repletion, cell numbers in thymus (DC and MC) and liver (DC) were greater than those of deficient littermates, but were less than those of controls (CC). Iron repletion postweaning reduced the cardiac hypertrophy (DC vs. DD and MC vs. MM) and increased splenic cell number compared to unrepleted deficient littermates (DC vs. DD). Thus, the severity and reversibility of impaired cellular growth is dependent on the timing and severity of the deficiency and the organ affected.

Lysozyme and other measures of immunity in young, mature, and aged rats.

Lysozyme, peroxidase, serum proteins, and immunoglobulins were measured in young (1 month), mature (13 months), and aged rats (25 months). Circulating levels of immunoglobulins G and A increased with maturity as did the globulin fraction of serum protein. Concentration of the bactericidal enzyme, lysozyme, was significantly increased in serum and kidney and significantly decreased in spleens of aged rats.