Potassium content of irradiated packed red blood cells in different storage media: is there a need for additive solution-dependent recommendations for infant transfusion?

Prevention of transfusion-associated graft versus host disease (TA-GVHD) by gamma irradiation is known to induce increased K+ in supernatant of packed red blood cells (PRBCs) stored in CPDA-1 and SAGM conservative solutions. However, no data exist for PRBCs in AS-3 medium which is considered safe for neonatal transfusion. We evaluated haemolysis and K+ release from irradiated AS-3 PRBCs and compared our results with reported data for SAGM and CPDA-1 PRBCs. Our results indicate that irradiated PRBCs stored in AS-3 after more than 7 days post-irradiation should not be used in massive and/or rapidly infused transfusions in neonates and infants.

[Transfusion-related acute lung injury (TRALI): emerging or main transfusion hazard?]. / Le syndrome respiratoire aigu post- transfusionnel (TRALI): complication émergente ou principale des transfusions?

Transfusion-Related Acute Lung Injury or TRALI is one of the most frequent severe transfusion reactions ( one third of Non Hemolytic Febrile Transfusion Reactions). It presents as a generally mild form of acute respiratory distress syndrome occurring 1 to 2 hours after transfusion of a plasma-containing blood component. This transfusion reaction appears, most of the time, in patients with focal inflammation and is of good prognosis when adequately diagnosed and treated. TRALI prevention rests on donor selection for plasma donation, hemovigilance system effectiveness and restrictive transfusion strategy.

Incidence of hantavirus infections in Belgium.

Over the last two decades, and from the moment that serological detection was possible, human hantavirus infections have been documented in most European countries. This paper summarises the available data on hantavirus cases in Belgium. These data enable the demonstration of the existence of a 3-year epidemic cycle in Belgium, which is apparently linked to rodent population dynamics.

Fast Multiplex polymerase chain reaction on boiled clinical samples for rapid viral diagnosis.

An assessment of optimal conditions for rapid simultaneous amplification of multiple human papillomavirus (HPV) sequences has been made using Thermus aquaticus DNA polymerase. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of well characterized cell lines. In our hands, Fast Multiplex PCR (FM-PCR), the technique of running multiple PCR reactions simultaneously with minimum incubation time at each temperature, was highly sensitive (amplification factor = 5 x 10(9) after 50 cycles), specific (100%) and reproducible (100%) for several microbiological applications. Diagnosis was generally obtained in less than 5 h after sampling. The results show that, after optimization of assay conditions, efficiency and specificity of Multiplex PCR depends exclusively on the primers design and concentration of the primers.

Suppression of the inhibitory effect of denatured albumin on the polymerase chain reaction by sodium octanoate: application to routine clinical detection of hepatitis B virus at its infectivity threshold in serum.

Ten to fifteen percent of posttransfusion viral hepatitis cases are still caused by HBV despite mandatory third generation screening procedures for HBsAg. There is thus an urgent need for a simple, time-cost-effective, but very sensitive test for routine HBV DNA detection in serum. Nested-primed PCR has been shown to detect purified HBV DNA at its infectivity threshold in serum. Since this is too labor-intensive for routine testing, we assessed the efficiency of a Fast PCR procedure, of three pairs of primers, and of thirty-five simple serum pretreatments with the aim to achieve the same sensitivity level. Using ten-fold dilution in phosphate buffered saline as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at the 2 x 10(3)/ml level in serum. Using either NaOH denaturation or sodium octanoate thermoprotection as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at its infectivity threshold in serum, while the classical phenol/chloroform/isoamylic alcohol/isopropanol/ethanol DNA purification procedure enabled us to reach the 10 virus particles/ml level. These results suggest that denatured albumin is responsible for the well known inhibitory effect of serum proteins on Taq polymerase. Because of its simplicity and its lower risk of sample-to-sample cross-contamination, the sodium octanoate thermoprotection method was chosen for routine clinical detection of HBV in serum. The clinical usefulness of this approach is demonstrated by the results obtained with HBsAg-negative acute hepatitis B incubation sera and with anti HBe-positive chronic hepatitis B sera.

Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements.

We evaluated minimal residual disease (MRD) in 23 CD5 + B-chronic lymphocytic leukemia (CLL) patients who achieved clinico-hematological remission confirmed by bone-marrow biopsy. MRD was evaluated by dual marker analysis flow-cytometry using CD5 and CD19 markers, and by the study of Ig heavy chain gene rearrangements using the fast polymerase chain reaction (PCR). According to our laboratory conditions patients were considered to be in complete phenotypic remission when total CD19+ cells were < 25% and the ratio of CD5 + CD19 + /CD19 + cells was < 25%. According to these strict criteria only 9 of the 23 patients were in complete phenotypic remission. In order to evaluate the sensitivity of the above method, PCR analysis of the configuration of the Ig heavy chain gene region was performed in 12 of these patients. Five of 7 patients in complete phenotypic remission retained a detectable monoclonal rearrangement of the Ig heavy chain gene. For the remaining 5 patients in partial phenotypic remission, only one failed to show a monoclonal band and this is probably explained by the presence of an unusual gene rearrangement. In conclusion, this study suggests that PCR is more sensitive than dual marker flow-cytometry for evaluation of residual disease and that it is indeed possible to achieve complete remission at the molecular level, in B-CLL. Nevertheless, we suggest a word of caution as this was a retrospective study, and samples were not assessed before treatment. Thus the possibility that apparent molecular remission might correspond to unusual gene rearrangements cannot be completely excluded in these cases.

Distribution of hantavirus foci in Belgium.

During 1999 and 2000, we performed rodent captures on 15 sites all over Belgium to evaluate the presence of hantaviruses in local rodent populations. Viral antibody and RNA detection was performed by ELISA/focus reduction neutralisation test and RT-PCR, respectively. We found hantavirus-positive rodents on 13 out of 15 trapping sites and 3 rodent species were found positive for hantavirus infection. Apart from Puumala virus that was carried by Clethrionomys glareolus, 2 additional rodent species, Microtus arvalis and Apodemus sylvaticus, were found antibody- and/or RNA-positive.

Human Granulocytic Ehrlichiosis in Belgium: an underestimated cause of disease.

OBJECTIVES: Human Granulocytic Ehrlichiosis (HGE) is a recently discovered zoonosis and, in Europe, not always included in laboratory testing when a patient presents with a history of tick bite. The available serology results indicate that HGE should be included in the screening panel when a tick-borne disease is suspected. METHODS: Serological methods were applied; i.e. indirect immunofluorescence and Western Blot analysis. Sixty-five serum samples from 47 patients were analysed, of six patients sequential samples were available. RESULTS: 33.8% of the submitted samples were found positive in indirect immunofluorescence, Western Blot confirmed 46.1% of these positive samples. CONCLUSIONS: Although the causative agent and the vector for HGE, Ixodes ticks, are present in Belgium, serology for HGE is seldom solicited. Ehrlichiosis is apparently not always considered as a plausible or possible cause for illness, even when the patient presents with a history of tick bite. We present here a, true be it, incomplete picture of the present situation in Belgium, but nevertheless indicating that it is warranted to test patients with a history of tick bite not only for Lyme disease, but also for HGE.

HIV transmission by transplantation of allograft skin: a review of the literature.

The fear of human immunodeficiency virus (HIV) transmission by means of allograft skin has led to a cautious approach to allograft donor selection. However, no irrefutable diagnostic test exists to determine the possible presence of HIV at the time of donation. In order to find ways of improving HIV donor screening practices for skin banks, we review the presence of HIV in human skin, explore the possible transmission of HIV by transplantation of human allograft skin, and discuss the reliability of existing HIV tests. The use of the polymerase chain reaction (PCR) as a sensitive detection system for HIV infection of skin biopsies, in combination with conventional routine HIV blood screening tests; could lower the risk of transmitting HIV to severely burned patients.

Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium.

Seoul hantavirus (SEOV), carried by Rattus rattus (black rat) and R. norvegicus (Norway, brown rat), was reported to circulate as well as cause HFRS cases in Asia. As Rattus sp. are present worldwide, SEOV has the potential to cause human disease worldwide. In Europe however, only SEOV prevalence in rats from France was reported and no confirmed cases of SEOV infection were published. We here report genetic and serological evidence for the presence of SEOV virus in brown rat populations in Belgium. We also serologically screened an at-risk group that was in contact with R. norvegicus on a daily basis and found no evidence for SEOV infection.

Molecular and serological evidence for Anaplasma platys and Babesia sp. infection in a dog, imported in Belgium, from Southern Spain.

This case report describes a dog suffering from a co-infection with Babesia and Anaplasma parasites. Anaplasma platys was found to be responsible for the anaplasmosis by molecular biology techniques, while microscopical and serological evidence was found for a coexistent babesiosis, although this could not be confirmed by polymerase chain reaction. Moreover, the possible risk of import of exotic pathogens is highlighted.

High-risk genital papillomaviruses and degree of dysplastic changes in the cervix: a prospective study by fast multiplex polymerase chain reaction in Belgium.

The epidemiologic studies with the least selection bias do not support the hypothesis that HPV types 16 and/or 18 are strongly associated with cervical cancer. In this preliminary report, we describe our findings regarding type 16, 18, and 33 detection rates in 323 normal and 71 dysplastic or neoplastic cervical scrapes using fast multiplex PCR. This modified PCR technique has been shown to be the most sensitive, specific, and reproducible DNA detection method for large epidemiologic studies. The results indicate a high relative risk of increasingly severe cervical abnormality associated with the presence of high-risk HPV DNA. The analysis of the prevalence and age data according to CIN status by non-parametric statistic tests highlights the importance of other factors inversely correlated with age in the cervical transformation process.

Risk factors inducing the persistence of high-risk genital papillomaviruses in the normal cervix.

To evaluate the risk factors associated with persistence of human papillomaviruses (HPV) types 16, 18, and 33 in the normal cervix, a prospective study was carried out in Belgium of 323 women without cytological evidence of cervical intraepithelial neoplasia. Demographic and clinical data were obtained by interview, and HPV DNA was assayed in cervical-swab specimens using the Fast Multiplex Polymerase Chain Reaction-based screening and confirmatory tests. A multivariable linear regression model was constructed using four well-known risk factors: the use of an oral contraceptive which is either triphasic, or monophasic and containing ethynylestradiol in association with either norethysterone, or levonorgestrel, or lynestrenol, or gestoden, or estrogenic and containing estriol (P = 8 x 10(-5)), a positive history of genital herpes simplex virus (HSV) infection (P = 10(-4)), an age inferior or equal to 30 years (P = 0.012), and cigarette smoking (P = 0.020). Crude and adjusted relative risks were calculated for each HPV persistence predictor. The data and the results of the molecular biology of high-risk genital HPVs are consistent with the hypothesis that the use of an oral contraceptive containing simultaneously and continuously both a potent estrogen and a high activity progestative is necessary to enhance significantly HPV transcription. These observations are also consistent with the hypothesis that the oral contraceptives and HSV genital infection are responsible for HPV persistence in the normal cervix but not for HPV-induced cervical transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

PIP: This prospective study evaluated the risk factors associated with persistence of human papillomaviruses (HPVs) types 16, 18, and 33 in the normal cervix. Cervical specimens were obtained from 323 women attending the cervical cancer screening clinic classified as normal on the basis of exfoliative cytology. Demographic, sexual, behavioral, and clinical data were obtained by interview. Crude and adjusted relative risks were calculated for each HPV persistence predictor. The data and the results of the molecular biology of high-risk genital HPVs were consistent with the hypothesis that the use of an oral contraceptive containing simultaneously and continuously both a potent estrogen and a high activity progestative was necessary to enhance significantly HPV transcription. A relatively weak correlation was found for cigarette smoking. In addition, it is noted that several other risk factors associated with young age, such as a high number of sexual partners, is also a predictor of acquiring HPV.

Fast PCR-based quantitative detection of B-cell malignancies.

Available methods for the detection of minimal residual disease in hematologic malignancies are limited by their poor sensitivity and/or complexity. In order to avoid these drawbacks, we used the fast PCR technique to amplify the hypervariable chain-determining region 3 (CDR 3) of the human immunoglobulin heavy-chain gene in boiled marrow nucleated cells. This enabled us to detect malignant B-cells down to a dilution of 1 in 1,300 marrow nucleated cells within 7 hours of sampling. This new quantitative method should be useful for monitoring therapy and detecting early disease relapse in B-lymphoproliferative disease since it is 10 to 60 times as sensitive as Southern blotting.

Evaluation of a commercial enzyme immunomembrane filter assay for detection of respiratory syncytial virus in clinical specimens.

A commercial enzyme immunomembrane filter assay (EIFA) for respiratory syncytial virus (RSV) was compared prospectively with isolation in cell culture and an enzyme immunoassay. A total of 595 respiratory specimens, mostly from pediatric patients, were examined. The EIFA was 70.96% sensitive and 72.40% specific in comparison with cell culture. Results for 40 specimens (6.72%) were uninterpretable, mainly due to filtration difficulties. Twenty-one (25%) of 84 specimens whose results were initially considered false-positive were subsequently confirmed positive after a blocking test with bovine anti-RSV serum. On the basis of the total number of confirmed positive results, the sensitivity and the specificity of the test were 87.90% and 75.77%, respectively.

Seroprevalence of human granulocytic ehrlichiosis infection in Belgium.

In order to determine the prevalence of human granulocytic ehrlichiosis (HGE) in Belgium, the sera of 216 patients previously diagnosed with Borrelia burgdorferi infection were analysed for possible coinfection with the agent of HGE. For this purpose, an indirect immunofluorescence assay was applied, and positive results were confirmed by Western blot using a 44-kilodalton recombinant protein (rP44) specific for the agent of HGE. Sixteen of the 216 (7.4%) sera tested were positive for the HGE agent using indirect immunofluorescence assay, and seven (3%) of them were confirmed positive by Western blot. These data suggest the agent for HGE is present in Belgium and may cause coinfection in patients infected with Borrelia burgdorferi, as has been reported in the USA and elsewhere in Europe. This is the first report documenting the identification of this agent in Belgium.

Fast PCR based therapeutic follow-up of T-cell malignancies.

Available methods for the detection of minimal residual disease in T-cell malignancies are limited by their poor sensitivity and/or by their complexity. With the aim of avoiding these drawbacks, we used the Fast PCR technique in order to amplify V delta 1-(D delta 1)-(D delta 2)-J delta 1 and V gamma I family-J gamma junctional sequences from nucleated cells of boiled bone marrow. We were thus able to detect malignant T-cells down to a dilution of 1 in 665 nucleated marrow cells, in less than 4 hours after sampling. This new quantitative method is promising for monitoring therapy and detecting early disease relapse in T-lymphoproliferative disease, since it is 2 to 35 fold more sensitive than Southern blotting.

Quantitation of Pseudomonas aeruginosa in wound biopsy samples: from bacterial culture to rapid 'real-time' polymerase chain reaction.

STATEMENT OF FINDINGS: We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Prevalence of high risk genital papillomaviruses in the Belgian female population determined by fast multiplex polymerase chain reaction.

Because in situ/filter hybridisation is not sensitive enough and because classical polymerase chain reaction (PCR) protocols are generally not sufficiently reproducible and specific, there is little accurate information on the prevalence of human papillomaviruses (HPV) 16, 18, and 33 infections in women without dyskaryotic changes of the cervix. In our hands, our Fast Multiplex PCR protocol has always been the most sensitive, specific, and reproducible DNA detection assay in all the microbiological and haematological applications we attempted (Vandenvelde C, Verstraete M, Van Beers D [1990]: Journal of Virological Methods 30:215-228; Vandenvelde C, Scheen R, Corazza F, Van Beers D [1991a]: Journal of Experimental and Clinical Hematology 33:293-297; Vandenvelde C, Scheen R, Van Beers D, Fondu P [1991b]: Journal of Experimental and Clinical Hematology 30:25-29). Using this new technique, cervical scrapes from 336 Belgian women attending the cervical cancer screening clinic were examined for the presence of these three high-risk genital papillomaviruses. Positive results were confirmed using another set of HPV-specific primers. Exactly one sixth of our population was found positive for one or more of these HPVs. Types 33 and 16 were significantly more prevalent than type 18. The nonparametric statistical analysis of the data suggests that some risk factors such as particular sexual habits, that are inversely related to age, must exist.