Current concepts for quality assured long-distance transport of temperature-sensitive red blood cell concentrates.

BACKGROUND AND OBJECTIVES: The German Armed Forces Blood Service in Koblenz supplies red blood cell concentrates (RBCs) to military and civilian institutions at home and to field hospitals during peacekeeping operations abroad. During long-distance transport, blood products can be exposed to extreme environmental conditions or inappropriate handling, which may compromise product quality. MATERIALS AND METHODS: Different active and passive cooling systems, cooling elements, packaging material and data loggers were examined in a climate chamber. A number of techniques for measuring temperature were investigated in order to preserve the blood products' quality during transport, including some field tests with multiparametric data recording. RESULTS: Any kind of active cooling systems, conventional cooling elements and customary packaging material, as well as temperature-sensitive labels, minimum-maximum thermometers and intra-product measurement were found to be unsuitable for military requirement. The best results were obtained when the passively cooling RCB 25 transport box (Dometic) was used together with latent heat/cold storage elements (deltaT) and Junior data loggers (Escort). CONCLUSION: The elaborated protocol allows temperatures to be maintained between 2 and 6 degrees C as required by European guidelines for at least 36 h each and between 1 and 10 degrees C as required by German guidelines for at least 48 or 64 h at ambient temperatures between -10 and 40 degrees C. Preliminary results indicate that care must be taken concerning additional factors such as air pressure variation or vibration.

Plague and other human infections caused by Yersinia species.

With an estimated 100 million victims, pandemically and epidemically occurring plague has been looked upon as a classical scourge of mankind during the last two millenia. Without treatment at least 50% of the affected individuals die from infection with Yersinia pestis, a bacterium belonging to the family of Enterobacteriaceae. The disease takes a fulminant course. After an incubation period of 2-6 days, bubonic plague primarily attacks one group of lymph nodes. The onset of pulmonic plague, transmitted by droplet infection, takes place within several hours and causes bronchopneumonia. Early recognition facilitates a promising antibiotic therapy with tetracycline, streptomycin or chloramphenicol. Human beings acquire the bacteria through bites of fleas from domestic rats in densely populated cities of countries with low hygienic standards, or sporadically in the open country from infected wild rodents. Laboratory procedure includes microscopy supplemented by immunofluorescence and cultivation of the bacterium from clinical material. Direct serology and PCR result in a fast detection of specific antigens or nucleotide sequences. Determination of serum antibodies is principally used for epidemiological investigation. Today, physicians in the civilized western world lack experience for the recognition of plague, and analytical techniques for diagnosis are only available in some specialized laboratories. Yersiniosis becomes primarily manifest as gastroenteritis caused by Yersinia enterocolitica or as pseudoappendicitis caused by Yersinia pseudotuberculosis and requires antibiotics only in severe septic cases. Different extraintestinal symptoms may be observed in dependence on the patient's HLA type and gender. The ubiquitous germ is mainly transmitted by the fecal-oral route via infected domestic or farm animals and contaminated food. The relevant virulence factors are encoded on a 70 kB plasmid common to all Yersinia species and strains that are human pathogens. The most important tools for laboratory diagnosis are culture from suitable body fluids and serological detection of specific antibodies. The infection rate among healthy individuals in Europe in terms of percentage of elevated IgA or IgG titers has been quoted to be 3-40% in different investigations but does not significantly correlate to direct bacteriological detection.

Community acquired diarrhea--the incidence of Astrovirus infections in Germany.

Astroviruses are increasingly recognized as a cause of human gastroenteritis. Electron microscopy (EM) has been considered the "gold standard" method for diagnosis, but this approach is limited to a few laboratories. We evaluated a commercial enzyme immunoassay (EIA) (IDEIA Astrovirus, DAKO Diagnostika, Hamburg, Germany) for the direct detection of antigen in fecal samples. In comparison to EM, the assay scored 100% in sensitivity and specificity (n = 213; 26 positive samples) and reacted with strains representing all known serotypes. Over an 11-month period 4,211 stool samples from unselected German patients suffering from acute gastroenteritis were examined. Etiologically responsible microorganisms were found in 13.0% of cases, with astrovirus the third most common pathogen (1.2%) behind Salmonella spp. (2.9%) and Rotavirus (2.5%), representing 13.5% of all positive specimens. Norwalk-like viruses (NLV), fungi, and protozoa were not tested. In infants of < 2 years of age (n = 458) the incidence of astrovirus infection was significantly higher (2.8%) compared to children of 2-7 years of age (n = 578; 1.7%) and those of > 7 years of age (n = 3,175; 0.9%). The frequency revealed a peak in winter (mean November-February: 2.0% versus other months: 0.8%).

Clonal diversity of Actinobacillus actinomycetemcomitans isolates from young adults with minimal periodontal disease.

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with both early-onset periodontitis and adult cases refractory to conventional periodontal therapy, although the organism has also been shown to be widely distributed among dentate healthy individuals. The observed disease status may be associated with a variation in virulence of different strains or clones. The aim of the present study was to analyse genotype distribution as assessed by an arbitrarily primed polymerase chain reaction (AP-PCR) among 51 isolates of A. actinomycetemcomitans recovered from more than 200 young adult recruits with no or minor periodontal disease. In addition, isolates from 25 periodontitis patients as well as reference strains were genotyped. Primers amplifying (i) a specific sequence in the ltxA region, (ii) a specific 16S rRNA sequence and (iii) sequences in the leukotoxin promoter region were used to verify species identity of the strains. Three random oligonucleotide primers were employed to analyse genomic polymorphisms of the organism by means of PCR. A total of 19 genotypes could be distinguished, which were grouped by cluster analysis into 5 major clusters based on genetic similarity and a complete linkage sort. Whereas 3 clusters assembled A. actinomycetemcomitans genotypes isolated from both healthy subjects and periodontitis patients, one cluster containing 4 different genotypes exclusively comprised isolates from healthy or gingivitis subjects. Another cluster with 2 genotypes consisted of strains originating from periodontitis patients (p < 0.05). One strain characterized by a specific 530 bp deletion in the promoter region of the leukotoxin region was identified in a Ghanese patient with localized juvenile periodontitis. It was concluded that there is considerable clonal diversity of A. actinomycetemcomitans strains isolated from healthy or periodontally diseased subjects, and that genetically closely related groups might be associated with health or disease.

Absence of an especially toxic clone among isolates of Actinobacillus actinomycetemcomitans recovered from army recruits.

Actinobacillus actinomycetemcomitans is a major periodontal pathogen which is associated with early-onset and adult periodontitis. The organism has been shown to be widely distributed among dentate, healthy individuals as well. It has been demonstrated that A. actinomycetemcomitans shows great genetic diversity. An especially virulent clone of the organism (JP2-like) with a specific 530-base pair (bp) deletion in the promoter region of the leukotoxin gene has been isolated from localized juvenile periodontitis patients and related subjects of African and African-American origin. The aim of the present study was to examine the presence of this specific clone employing specific oligonucleotide primers in a polymerase chain reaction among 51 isolates of A. actinomycetemcomitans recovered from 201, 18- to 25-year-old recruits with no or minor periodontal disease. In addition, clinical isolates from 37 periodontitis patients were analyzed as well as reference strains ATCC 29524, NCTC 9710, Y4 and JP2. Primers amplifying a specific 285-bp amplification product in the ltxA region of the leukotoxin gene and a specific 547-bp amplification product of 16 S rRNA were used to genetically confirm identification of the organisms. Primers amplifying sequences in the leukotoxin promoter region were used to identify the deletion. Species specific primers definitively identified all A. actinomycetemcomitans isolates. No isolates from army recruits or the reference strains displayed the deletion in the leukotoxin promoter region. However, in the periodontitis group, a 24-year-old individual from Ghana with localized juvenile periodontitis was identified with an intraoral infection with highly toxic A. actinomycetemcomitans (JP2-like). Present results confirm previous observations of absence of a highly toxic clone of A. actinomycetemcomitans among periodontally healthy and diseased Caucasians as well as a possible presence in localized juvenile periodontitis in individuals of African origin.

Immunoblotting of Yersinia plasmid-encoded released proteins: a tool for serodiagnosis.

The plasmid-encoded, released proteins (RPs) of Yersinia enterocolitica serotypes 09 and 03 were separated by sodium dodecyl sulfate (SDS)-pore gradient gel electrophoresis. The RP-patterns of both serotypes proved to be identical. Five major proteins of M(r) 27,000, 34,700, 35,600, 45,800, and 46,800 were detected. Spontaneously plasmid-cured derivatives of the two serotypes lost the feature of protein release. By immunoblotting of RP with sera from patients suffering from acute Yersinia infections, specific and reproducible band patterns were obtained. Laser scan densitometry was applied to record the immunoreactions quantitatively. Predominant bands were detected at an M(r) of 34,700 and 35,600. IgA and IgM antibodies appeared as acute-phase markers rapidly decreasing in the reconvalescent phase. In contrast, immunoblots of patients with supposed chronic yersiniosis were characterized by a persisting IgA and elevated IgG reactivity. The application of RP as diagnostic antigens proved to be advantageous because they are naturally separated from cross-reacting proteins, common to pathogenic and nonpathogenic strains of Yersinia enterocolitica and Y. pseudotuberculosis.

Evaluation of an immunofluorescence assay for specific detection of immunoglobulin G antibodies directed against Helicobacter pylori, and antigenic cross-reactivity between H. pylori and Campylobacter jejuni.

An immunofluorescence assay (IFA) for the detection of immunoglobulin G antibodies directed against Helicobacter pylori was evaluated by comparing 20 serum specimens from patients with a positive urease test on biopsy material and 20 serum specimens from patients with a negative test and with defined clinical symptoms. The resulting anti-H. pylori titers were classified as follows: negative, less than or equal to 64; borderline, 128; and positive, greater than or equal to 256. By using these criteria, the IFA was subsequently tested, using 100 serum specimens from patients with gastric complaints. Overall, the titers were 71% positive, 10% borderline, and 19% negative. Depending on the patients' biopsy urease test results, the sensitivity and specificity of the assay were calculated to be 96%. Furthermore, these sera were classified into three subgroups on the basis of clinical manifestations: gastritis with 74% positive and 10% borderline titers, duodenal ulcer with 84% positive and 4% borderline titers, and gastric ulcer with 52% positive and 16% borderline titers. A serologic follow-up study was carried out with three patients with gastric ulcers who had been treated with colloidal bismuth subcitrate for 4 weeks and erythromycin for the final 2 weeks. The results indicate that a significant decrease in titer could be expected within 9 to 12 months after successful therapy, as determined by repeated negative CLO tests. Absorption experiments demonstrated that possible cross-reactivity between H. pylori and C. jejuni did not influence serodiagnosis.