Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

Bacterial screening of platelet concentrates: results of 2 years active surveillance of transfused positive cultured units released as negative to date.

BACKGROUND: The BacT/ALERT system for bacterial monitoring of platelet concentrates (PCs) was introduced in the Netherlands in 2001. Samples are cultured for 7 days, and as a result of the short shelf-life of PCs, they are usually released as 'negative to date'. Therefore, some of the PCs have already been transfused at the moment of a positive signal in continued cultures in the BacT/Alert. It is unclear, however, whether these PCs are associated with more transfusion reactions. METHODS: During a 2-year period clinical data were collected from all patients who received PCs released as 'negative to date' but with a positive bacterial culture after being transfused. RESULTS: Data of 158 patients who received PCs with confirmed positive bacterial culture tests were analysed. Two patients developed a transfusion reaction. In both PCs, Propionibacterium was cultured. The imputability as related to the transfusion was classified as unlikely in both patients. CONCLUSION: Two of 158 transfusions of PCs released as 'negative to date', but with a confirmed positive BacT/ALERT result, were initially associated with transfusion reactions. However, the imputability of both reactions was low.

[An outbreak of Q fever in The Netherlands--possible link to goats]. / Een uitbraak van Q-koorts in Nederland--mogelijk verband met geiten.

In 2007, 73 cases of Q fever were identified through reports and retrospective analyses; the affected region extended from Tilburg in the southwest to Arnhem in the northeast. The infections arose in late spring, particularly in May and June. Several spontaneous abortions due to Q fever occurred on 4 dairy goat farms in the same region. The national incidence of spontaneous abortion due to Q fever was 6 cases in 2006 and 7 in 2007. Climatically, this southern region was extraordinarily dry during April 2007. All pregnant women from a small region with the highest incidence in northeast North Brabant were called for diagnostic testing. Infected patients were followed for symptoms and ultrasound was performed as indicated. A definitive source of the infection could not yet be identified. Favourable climatic conditions were suspected as the cause for the combination of widespread dissemination among goats and transmission to humans. Q fever is a zoonosis caused by Coxiella burnetti, a microorganism dispersed in great numbers in the area in which an infected animal gives birth. C. burnetti is particularly resistant to chemical and physical factors and can disperse by air across large distances under dry climatic conditions. Q fever should be considered in patients in The Netherlands who present with lower airway infection and, in rare cases, hepatitis. Reporting atypical clusters ofpneumonia to the Municipal Health Service (GGD) is advisable. The GGD maintains close contact with Animal Health Services, which is aware of current infectious animal diseases. Targeted investigation can identify the source of infection and eliminate it. Greater awareness can prevent delays in diagnosis and treatment and help identify chronic forms at an early stage or prevent them.

Idiopathic intracranial hypertension without papilledema.

We describe 10 patients with idiopathic intracranial hypertension who did not have papilledema. Idiopathic intracranial hypertension without papilledema, although rarely reported, may well be a clinically important headache syndrome. Historical and demographic features of patients with idiopathic intracranial hypertension without papilledema are similar to those of patients with papilledema. Obese women with chronic daily headache and symptoms of increased intracranial pressure, pulsatile tinnitus, history of head trauma or meningitis, an empty sella on imaging studies, or a headache that is unrelieved by standard therapy should have a diagnostic lumbar puncture. Findings from laboratory and neurologic investigations are normal in most patients with idiopathic intracranial hypertension without papilledema. Initial management should include removal of possible inciting agents, weight loss if applicable, and standard headache therapy. Lumbar puncture and diuretic therapy should precede a trial of corticosteroids. Surgery (lumboperitoneal or ventriculoperitoneal shunt or perhaps optic nerve sheath fenestration) may be indicated for prolonged incapacitating headache that is not responsive to medical management or lumbar puncture.

[Rat bite fever after a bite from a tame pet rat]. / Rattenbeetziekte na een beet van een tamme huisrat.

A 43-year-old woman presented with a generalized febrile illness, an exanthema with mixed maculopapulous and pustulous eruptions on the lower halves of the extremities, elbows, knees, palms and soles. There was also severe arthralgia and asymmetric arthritis. The diagnosis was rat bite fever. The disease became manifest eight days after she was bitten by a pet rat. Rat bite fever can easily be missed, even after adequate anamnesis and physical examination, while the differential diagnostic considerations are numerous. Our patient was cured completely after intravenous administration of penicillin G. Antimicrobial therapy was completed by an oral course of doxycycline.

[Histoplasma capsulatum infection, a manifestation of AIDS unusual for The Netherlands]. / Histoplasma capsulatum-infectie, een voor Nederland ongewone manifestatie van de ziekte AIDS.

The history of 29-year-old male from Surinam with antibodies to HIV-1 and long-lasting fever, lymphadenopathy, pain in the right upper abdomen and a granulomatous hepatitis is described. The patient suffered from disseminated histoplasmosis, a fungal disease rare in The Netherlands, which is the indicator disease for the diagnosis of AIDS (CDC-IVCI). It is stressed that in seropositive patients coming from endemic areas, including Surinam, the possibility of this disease should be considered.

Spontaneous low cerebrospinal fluid pressure headache.

A 69 year old man presented with headache on arising and relieved with recumbency. His clinical presentation, laboratory data, response to treatment and radiographic evaluation were consistent with spontaneous low cerebrospinal fluid pressure from CSF hyperabsorption. Cisternography showed rapid accumulation in the kidneys and urinary bladder, without evidence of CSF leak. His headache was eventually controlled with two blood patches and oral caffeine.

Quantitative gas chromatography of Bacteroides species under different growth conditions.

From 56 strains of strictly anaerobic gram-negative rods isolated from stool and purulent lesions the fermentation products in the presence and absence of hemin were determined by quantitative gas-solid chromatography, using a simple and more rapid chromatographic procedure. With hemin the fermentation products were propionic, acetic, lactin and succinic acid. Without hemin no or little succinic acid was formed and the main products were lactic and acetic acid. In both groups the distribution of subspecies was determined and the production of fatty acids measured quantitatively. Fourteen strains of the lesion group showed a higher metabolic activity, resulting in an increased total acid production caused by an excessive production of acetic and lactic acid. This characteristic is probably a virulence factor in these strains. All strains were protoporphyrin- and oxgall-dependent. It is postulated that these substances are used for the production of cytochromes which permits the formation of succinic acid by a fumarate reductase resulting in an increased growth rate and growth yield.

Headache associated with aseptic meningitis.

A retrospective analysis of all patients admitted with the diagnostic codes of aseptic or viral meningitis was performed at two institutions over 3 years. Forty-one patients with cerebrospinal fluid confirmation of aseptic meningitis (increased protein; increased white count; negative gram stain; and negative fungal, tuberculosis, and bacterial cultures) were analyzed. All the patients had headache, which was typically severe and bilateral in 39 of the 41 patients. The headache was of abrupt onset or the worst of the patient's life in 24 of the patients. The quality of the headache, when described, was usually throbbing (11 of 14). Nineteen patients had prodromal symptoms, including malaise, myalgia, gastrointestinal symptoms, and urinary tract infections. All had associated symptoms, including nausea (25), vomiting (23), photophobia (18), stiff neck (25), and back pain (11). Thirty patients were febrile. Lumbar puncture was performed for headache and fever unexplained by systemic illness in 30 patients, meningeal signs in 15, headache of abrupt onset or the worst headache ever in 24, neurologic signs or symptoms in 12, and for other reasons in 2. Computerized tomography, when performed, was negative in all cases. Focal neurologic findings were present in 5 patients, a decreased level of consciousness in 6, and papilledema in 1. A severe headache that worsens, is abrupt in onset, or is the worst of the patient's life could be due to aseptic meningitis, bacterial meningitis, or a subarachnoid hemorrhage. Although not universally present, meningeal signs, fever, and neurologic signs or symptoms should alert one to a possible central nervous system infection.

Antibodies raised against rough mutants of Escherichia coli and Salmonella strains are opsonic only in the presence of complement.

The opsonic capacity of antisera raised in rabbits against rough (R) mutants and smooth (S) parent strains of Escherichia coli and Salmonella typhimurium were studied. All specific antibodies in the antisera belonged to the immunoglobulin G (IgG) class. Radioactively labeled bacteria were preincubated in various dilutions of antisera, in which complement was inactivated. Fresh normal rabbit serum, as a standard complement source, was used in some experiments. After preincubation, washed bacteria were added to normal human neutrophils. Opsonization of R mutants for 5 min in 5% fresh normal rabbit serum resulted in effective phagocytosis; S strains needed at least a 30-min opsonization time or 20 to 50% serum. After incubation for 5 min in diluted, homologous antisera, phagocytosis of S strains was optimal, but preincubation of R mutants in diluted, homologous antisera did not lead to amelioration of phagocytosis compared with that of bacteria preincubated in buffer only. However, when fresh normal serum was added to homologous antisera, uptake of R mutants occurred at a faster rate than that of bacteria opsonized in fresh serum alone. Using six clinical isolates of members of the family Enterobacteriaceae, we found that, with or without complement, antisera raised against E. coli J5 or S. typhimurium Re had, with the exception of one strain, no opsonic activity for these strains. Thus, the protective effect of R antisera in gram-negative bacteremia, as shown by several investigators, is unlikely to be mediated through enhanced opsonization of invading bacteria by IgG antibodies directed against these R mutants.

Determination of the degree of bacterial contamination of whole-blood collections using an automated microbe-detection system.

BACKGROUND: The prevalence of bacterial contamination in whole-blood collections, either with immediate sampling or sampling after overnight storage as whole blood at 20 degrees C, is determined. STUDY DESIGN AND METHODS: Whole blood was collected under blood bank conditions in special five-bag systems, allowing sampling in a closed system for culture bottles. Samples were taken within 2 hours after collection (Group 1) or after overnight storage of the whole blood at 20 degrees C (Group 2). Culture bottles were incubated for 7 days, and positive samples were entered on agar plates for confirmation and determination. RESULTS: In Group 1, 9219 units were tested; 27 units were positive with positive subculture, that is, 0.29 percent with a 95% CI of 0.19 to 0.42 percent. In Group 2, 9038 units were tested; 36 units were positive with positive subculture, that is, 0.39 percent with a 95% CI of 0.28 to 0.55 percent. No significant difference could be found between the two test groups. The majority of bacteria were either Staphylococcus (all coagulase-negative) or Propionibacterium species. CONCLUSION: For a total of 18,257 units, 0.34 percent (CI, 0.25-0.44) of whole-blood collections appeared to have bacterial contamination (mainly skin-derived). Overnight storage of whole blood at 20 degrees C did not have a significant effect on the prevalence of bacterial contamination.

Sterility testing of blood products in 1994/1995 by three cooperating blood banks in The Netherlands.

BACKGROUND AND OBJECTIVES: Dutch regulations require blood banks to check the sterility of random blood components to detect contamination during preparation. MATERIALS AND METHODS: We reviewed the results of two years' testing, using standard bacteriologic methods. RESULTS: Of all tested components, 0.5% were contaminated, with Staphylococcus epidermidis being the most frequently detected microorganism. Platelet concentrates showed higher rates of contamination, especially when pooled. Leukocyte-depleted red cell concentrates showed much lower contamination than red cell concentrates that had not been leuko-depleted. CONCLUSIONS: The rate of contamination compares well with that reported by others in the literature. Since most contamination occurs from the phlebotomy site, most of the bacteria detected were derived from the skin. Leukocyte reduction lowers the rate of contamination.

Intracellular polysaccharide of Bacteroides fragilis.

Formation of iodophilic polysaccharide (IPS) from glucose was demonstrated in 27 strains of Bacteroides fragilis. Synthesis was dependent on the glucose concentration of the medium, the pH and the growth phase. When glucose was in short supply the cellular polysaccharide was degraded rapidly at pH 4.5 to 6.5 and fatty acids accumulated in the medium. Storage of IPS was not responsible for the low carbon recoveries observed in fermentation balance studies. In electron micrographs of thin sections, the IPS was observed as cytoplasmic granules dispersed throughout the whole cell. After extraction and purification the IPS was characterized as a glycogen.

Semielectronic turbidimeter for automated monitoring of bacterial growth in test tubes.

An automated turbidimeter for measuring bacterial growth in ordinary test tubes is described. The device records and prints adsorbance, expressed as Klett units, of 60 cultures every 15 min. Provision is made for either aerobic or anaerobic incubation. The device is adaptable to modification, depending upon local requirements and availability of computation facilities.

Iron requirement and chelator production of staphylococci, Streptococcus faecalis and enterobacteriaceae.

The effect of iron deprivation on growth of 101 aerobic strains of gram-positive and gram-negative bacteria was studied on agar media in the presence of various concentrations of the synthetic iron chelator ethylene diamine diorthohydroxyphenyl acetic acid (EDDA) and the iron binding protein transferrin. Growth of Staphylococcus epidermidis was inhibited by 15 mM EDDA and 1.5 mM transferrin. Staphylococcus aureus was only inhibited by 44 mM EDDA and not by transferrin. None of the strains of S. faecalis was inhibited. The majority of the enterobacteriaceae (E. coli, Salmonella spp, Klebsiella spp) was inhibited by 44 mM EDDA and 1.5 mM transferrin. The relation between susceptibility and concentration of EDDA and transferrin was expressed as S-value for each species. Iron supply with various iron compounds could restore the effects of inhibition. In all species except in S. faecalis iron chelator production could be demonstrated, using indicator plates of media containing EDDA and flooded with 10(4)--10(5) colony forming units of indicator organisms. The iron chelator of both S. epidermidis and S. aureus could stimulate growth of S. epidermidis, but not that of enterobacteriaceae. Iron chelators from all gram-negative bacteria were functionally interchangeable, but did not stimulate growth of gram-positive bacteria.

A new selective medium for the culture of clostridia from human faeces.

A new selective medium, sulphite-polymixin-milk (SPM) agar, for the isolation of clostridia from faeces is described. This medium contains whole cow's milk, which favours the growth of clostridia over that of Bacteroides and Bifidobacterium spp., and only small amounts of colistin (10 micrograms/ml) for suppressing growth of coliforms. The alkaline end-products of clostridia give rise to yellow colonies on a red background which are easy to distinguish from the red colonies of other not completely inhibited bacteria. The medium is suitable for isolation of many clostridia species. Comparisons were made between SPM and several other media for recovery of clostridia. The detection limit on SPM agar was more than 10(3) below that on Reinforced Clostridia Agar (Oxoid CM101).

Diversion of first blood volume results in a reduction of bacterial contamination for whole-blood collections.

BACKGROUND AND OBJECTIVES: In a previous study we established a reliable setpoint for the prevalence of bacteria in whole blood. In the present study we investigated the possible preventive effect, of diversion of the first 10 ml of a blood donation, on the bacterial contamination rate. MATERIALS AND METHODS: To divert the first 10 ml of a whole-blood donation, we used a special five-bag system equipped with a Composampler device. After venepuncture, the first 10 ml of a donation was sampled into a vacutainer tube. This was followed by the collection of the whole-blood unit. The extra bag allowed direct sampling of the final donation in a closed system for BacT/Alert. Whole-blood samples were taken after storage (2-14 h at 20 degrees C) and subsequent mixing. BacT/Alert culture bottles were incubated until positive, or for 7 days if negative. Confirmation and identification of positive cultures was performed according to internationally recognized standard reference methods. RESULTS: The prevalence of bacteria in whole blood, as determined by using standard collection techniques, was 0.35% (95% confidence interval 0.27-0.44%, n = 18 257). After diversion of the first 10 ml this value was significantly lower: 0.21% (P < 0.05, 95% confidence interval 0.12-0.35%, n = 7087). Most strikingly, a reduction in the frequency of staphylococcal species was observed (P < 0.02, reduction from 0.14 to 0.03%). CONCLUSIONS: Diversion of the first 10 ml of blood was shown to contribute significantly to a reduction in the prevalence of superficial skin bacteria in whole-blood units. In our opinion, blood collection systems should be adapted to use the first 10-30 ml of a whole-blood donation for testing purposes.