Anti-leucocyte antibodies in platelet apheresis donors with and without prior immunizing events: implications for TRALI prevention.

BACKGROUND AND OBJECTIVES: Transfusion-related acute lung injury (TRALI) prevention strategies in platelet (PLT) apheresis donors focus on identifying antileucocyte antibody-positive donors. The use of microbead based assays for screening purposes is hampered by the lack of a consensus cut-off for TRALI prevention and the undefined role of anti-leucocyte antibodies in never-alloexposed donors. This study evaluated anti-leucocyte antibody assays in PLT apheresis donors with and without prior immunizing events with special focus on microbead assay cut-offs, antibody specificities and their potential significance in never-alloexposed donors. MATERIAL AND METHODS: Blood samples of male and female PLT apheresis donors with and without history of prior immunization were tested for anti-leucocyte antibodies. RESULTS: Of 262 female and 118 male PLT apheresis donors, 37·4% had prior immunizing events. Fifty-eight of 238 (24·4%) donors without prior immunizing event had anti-HLA antibodies confirmed in microbead single antigen assay (mean fluorescence intensity (MFI) >500). Even with a cut-off MFI >3000, anti-HLA antibodies were detected in 10·6% of female and 4·3% of male donors without history of immunization. Of the antibody specificities found, 6 of 17 (35·3%) anti-HLA-A, 4 of 8 (50·0%) anti-HLA-B and 4 of 6 (66·6%) anti-HLA class II antibodies have been detected in donors associated with TRALI cases in the literature. CONCLUSION: Platelet apheresis donors without history of immunization have anti-leucocyte antibodies that potentially can cause TRALI. In our opinion, this cohort should be included in screening strategies for TRALI prevention. As references and consensus cut-offs have not yet been established, it is premature to use microbead assays as standard for donor screening.

Fatal outcome of a hepatitis B virus transfusion-transmitted infection.

BACKGROUND AND OBJECTIVES: In 2008, hepatitis B virus (HBV) DNA testing was not yet mandatory for the screening of blood donations in Switzerland. At that time, HBsAg was the only specific mandatory marker for HBV. The importance of high sensitivity for HBV NAT screening is shown. MATERIALS AND METHODS: Donor and recipient of a transfusion-transmitted HBV infection were followed up. Multiple samples were tested for HBV serological and molecular markers. RESULTS: At donation, the donor appeared healthy, HBsAg was negative and had a normal ALAT level. Ten weeks later, clinical symptoms suggested acute HBV infection as was confirmed with positive HBsAg, HBeAg, anti-HBc IgG, anti-HBc IgM and anti-HBe. The archived sample from the original donation was negative for anti-HBc, but positive for HBV DNA (17 IU/ml). A recipient transfused with the red cell concentrate was HBV DNA positive (3100 IU/ml) 3 months post-transfusion. After five months, HBsAg, HBeAg, anti-HBc and HBV DNA (1.1 x 10(11) IU/ml) were positive. Two weeks later, the patient died from complications associated with HBV infection and his underlying bone marrow disease. CONCLUSIONS: The present case illustrates the importance of introducing highly sensitive HBV NAT screening strategy to prevent possible HBV transfusion-transmitted infections from donors with low viral load.

The risk of a second diagnostic window with 4th generation HIV assays: Two cases.

BACKGROUND: Despite the improved sensitivity of the 4th generation combined antigen/antibody HIV assays, detection of HIV in the early phase of an infection may still be ineffective. OBJECTIVES: Description of two cases that highlight the existence of the "second diagnostic window phase" observed with commonly used sensitive 4th generation HIV assays. STUDY DESIGN: Samples were screened with different 4th generation HIV assays. HIV infection was confirmed with an HIV I/II antibody assay, a HIV-1 p24 antigen assay, the INNO-LIA HIV I/II Score Line immunoassay and HIV-1 PCR. RESULTS: In both investigated cases, the limitations of the 4th generation HIV assays within the second diagnostic window were apparent. CONCLUSIONS: The overall sensitivity of the commercial 4th generation HIV assays is currently higher than the 3rd generation HIV assays. Nevertheless, the rare occurrence of a second diagnostic window with 4th generation HIV assays strongly suggests that the following up testing algorithms need to be adjusted accordingly.

Molecular basis of the Rh antigen RH48 (JAL).

BACKGROUND AND OBJECTIVES: RH48 (JAL) is a low-incidence Rh antigen of unknown molecular background associated with weakened expression of RhCE antigens. The objective of this study was to establish the molecular basis of JAL. MATERIALS AND METHODS: Seventeen JAL+ samples, from seven black (one of them a Brazilian of mixed race: black/Caucasian), nine European Caucasians and one Asian individuals, were typed with anti-D, -C, -c, -E and -e. Some samples were also tested for V/VS and ce (f). Titration studies and flow cytometry were used to analyse the expression of the JAL antigen and genomic DNA sequencing of all RHCE exons was conducted on all samples. Routine genotyping for RHCE was carried out on all samples. Screening of RHD exons 1-10, which included detection of the DAU allele, was carried out on all except one of the black samples. The Caucasian samples and remaining black sample were screened for the DAU mutation 1136C>T (T379M). RESULTS: Six black individuals had the Dce haplotype with RHCE mutations 340C>T (R114W) and 733C>G (L245V) [V/VS] and the RHD mutation T379M [DAU]. One mixed race individual had the Dce haplotype with the RHCE mutation 340C>T (R114W) but without the V/VS or DAU mutation. Eight Caucasians had the DCe haplotype with the 340C>T mutation. One Caucasian and one Asian had the Dce haplotype with a different mutation in an adjacent nucleotide, 341G>A (R114Q). All Caucasian individuals were negative for the DAU mutation 1136C>T (T379M). Previously described weakness of CE-related Rh antigens when present in single dose on JAL+ samples of DCe and Dce haplotypes was observed. Weak expression of V/VS was observed in the three black samples tested and weakness of JAL was observed in the black samples compared to the Caucasian samples. CONCLUSION: The same mutation (340C>T, R114W) in two different haplotypes (DCe and Dce) and another mutation (341G>A, R114Q) in one of these haplotypes (Dce) are associated with expression of the JAL antigen. One of the RHCE mutations detected in our samples (340C>T) has been previously described but not in association with the JAL antigen. Our results indicate that the previously described RhCeMA and ce(s)(340) alleles encode the JAL antigen. Expression of V/VS antigen is weakened in the presence of JAL and expression of JAL is usually weaker when associated with the Dce haplotype compared to DCe.

Incidence of viral markers and evaluation of the estimated risk in the Swiss blood donor population from 1996 to 2003.

Among the well known transfusion-associated risks, the transmission of pathogenic viruses is regarded as one of the most serious. Over the past two decades, a series of overlapping safety procedures have been successively implemented to minimise this risk. It is now generally considered that the risk of transmitting viral infections via blood products is very low in developed countries. The present study analyses the incidence of the key infectious diseases HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) between 1996 and 2003 from 99% of voluntary repeat blood donors visiting the blood transfusion service of the Swiss Red Cross. Furthermore the estimated risk of these viral markers was calculated. From 1996 to 2003 the incidence rate for HCV decreased continuously, whereas no significant decrease in the incidence rate of HIV and HBV was observed. From 2001 to 2003, the last calculated period, the residual risk was estimated to be 1 in 1,900,000 for HIV, 1 in 2,200,0000 for HCV and 1 in 115,000 for HBV, respectively. This agrees with international studies, which have been shown that the estimated residual risk for HBV between 1996 and 2003 is higher than that of HCV and HIV.

Improved sensitivity of the Chlamydia trachomatis Cobas Amplicor assay using an optimized procedure for preparation of specimens.

The aim of this study was to evaluate the performance of an improved sample preparation procedure that enhances the sensitivity of the Chlamydia trachomatis Cobas Amplicor assay (Roche, Switzerland). This procedure was developed after it was observed that, in some cases, endocervical swabs positive for Chlamydia trachomatis in a direct immunofluorescence assay were negative in the Chlamydia trachomatis Cobas Amplicor. For this procedure, the initial sample volume was increased over that recommended by the manufacturer, the sample was concentrated by centrifugation, and the yield of the extraction was increased by an additional enzymatic lysis. Five hundred sixteen endocervical swabs from women visiting a family planning clinic were tested in parallel by the Chlamydia trachomatis Cobas Amplicor using the extraction procedure recommended by the manufacturer and the modified in-house sample preparation procedure. Eight samples were positive with both procedures. Seven additional samples were positive with the in-house method only. The results show that the in-house sample preparation procedure markedly improves the test sensitivity of the Chlamydia trachomatis Cobas Amplicor assay. Consequently, the use of this improved screening protocol will facilitate the detection of even low-level infections with Chlamydia trachomatis, which in turn will lead to the introduction of earlier therapeutic interventions.

Hepatitis C virus-polymerase chain reaction minipool testing: 3 years in the largest Swiss blood transfusion service.

BACKGROUND AND OBJECTIVES: Hepatitis C virus-polymerase chain reaction (HCV-PCR) minipool testing can improve the safety of labile blood products owing to a reduction in the diagnostic preseroconversion window period. In Switzerland, HCV-PCR minipool testing for the release of labile blood components became mandatory in September 1999. In the largest Swiss blood transfusion centre, HCV-PCR minipool testing began in January 1999. This report analyses the performance of the test during a 3-year period: 1 January 1999 to 31 December 2001. MATERIALS AND METHODS: EDTA-blood was collected in either standard tubes or plasma preparation (PPT) tubes from 10 blood transfusion services in Switzerland and then sent to the Blood Transfusion Service SRC Berne. Up to 48 donor samples were pooled overnight using Tecan Genesis RSP 200/8 pipettors. Viral RNA was extracted by using the Qiagen QIAamp 96 viral RNA BioRobot kit on a BioRobot 9604. For PCR amplification and detection of HCV or internal control (IC) sequences, the Roche Cobas Amplicor v2.0 test kit was used. Data management, pool resolution and identification of positive samples were performed using the PMS Software from Tecan. RESULTS: In the 3-year period from 1 January 1999 to 31 December 2001, 839056 blood donor samples were tested in minipools of up to 48 samples. Thirty-five HCV-PCR-positive donations were identified. Thirty-four samples had antibodies against HCV and were therefore also detected by screening for antibody to HCV (anti-HCV). In October 2001, one seronegative (but PCR-positive) donor was detected. CONCLUSIONS: HCV-PCR minipool testing was successfully introduced in the largest Swiss blood transfusion service. It was shown that the release of HCV-PCR minipool results can be accomplished concurrently with the results of serological analysis. The challenge with a seronegative, but PCR-positive, donor demonstrates that the minipool testing strategy adds additional safety to blood products.

Detection limit, cut-off and specificity of an improved rapid assay for cardiac troponin T.

The performance of an improved version of the troponin T rapid test TROPT Sensitive was investigated in a multicentre evaluation at twelve centres. The detection limit and the cut-off were determined in a method comparison with Elecsys Troponin T using a total of 365 samples from patients with suspected acute coronary syndromes and 91 samples from healthy blood donors or non-cardiological patients. The analytical specificity was determined by measuring 1271 blood samples from blood donors without any myocardial injury. The test cut-off (90% of results positive) is 0.08 microg/L, and the detection limit is about 0.05 microg/L. The analytical specificity of the test is between 99.7 and 99.9%. With its small area of undefined significance between positive and negative results and its high sensitivity and specificity, TROPT Sensitive is very well suited to the reliable detection of troponin T positive patients with acute coronary syndromes.

Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum beta-lactamases.

The aim of this study was to detect point mutations in extended-spectrum beta-lactamase (ESBL) genes in a background of wild-type (non-ESBL-producing) bacteria using a highly sensitive and specific method developed for this purpose. The ligase detection reaction-polymerase chain reaction (LDR-PCR) method was used to test different ESBL-producing strains and clinical isolates for a specific point mutation in the bla SHV-ESBL gene (glycine to serine mutation at position 238) and was compared with the commercially available E test ESBL (AB Biodisk, Sweden). Nine of the 40 clinical isolates tested were positive for the bla SHV-ESBL point mutation when tested by the LDR-PCR method but negative when tested by the E test. In contrast to the E test or other molecular genetic tests, the LDR-PCR method is able to identify a single bacterium with a point mutation in a background of 100,000 wild-type (non-ESBL-producing) bacteria.

[Screening of Chlamydia trachomatis: is the diagnostic efficacy good enough?]. / Chlamydienscreening: Ist das Nachweisproblem gelöst?

OBJECTIVE: In the background of discrepant results of C. trachomatis testing in women with suspected genital infections, three different tests have been compared. METHOD: 594 endocervical swabs were collected from women visiting the family planning clinic in Aarau. Each of the following tests was evaluated: Cobas Amplicor Chlamydia trachomatis, IF Chlamyset, and Chlamydia IF direct. If one of these three tests was positive, the fixed material on the slides was scraped off, and a PCR was performed. RESULTS: 15 out of 594 samples were "true'-positive. PCR recognized 14 of them, the IF Chlamyset 13, and the Chlamydia IF 8. With the IF Chlamyset 37 samples were false-positive. CONCLUSIONS: PCR testing exhibits a good sensitivity and a high specificity, but we think that the sensitivity can be increased by improving the sample preparation step. The Chlamydia IF direct has a good specificity, but a rather low sensitivity. It should be noted that the PCR has the potential to become a new gold standard for the detection of C. trachomatis.

Polymerase chain reaction (PCR) for detection of pathogenic microorganisms in bacteriological monitoring of dairy products.

The presence of pathogenic bacteria poses a serious problem in sustaining the safety of dairy products. Microbiological routine controls of these products make use of selective culture techniques. To detect pathogenic species, isolated colonies are characterized by specific metabolic activities and by serotyping. We present an alternative biochemical approach that does not require culture of bacteria. The total bacterial populations of food samples were isolated by centrifugation and analysed by PCRs specific for pathogenic species. A total of 90 raw milk samples and dairy products made from raw milk were screened by this method for the presence of Listeria monocytogenes, Escherichia coli, enterotoxigenic E. coli, Campylobacter jejuni and C. coli. Detection rates were 12/90 (13%) for L. monocytogenes, 41/90 (46%) for E. coli, 18/90 (20%) for enterotoxigenic E. coli producing heat-labile toxin type I or heat-stable toxin type I, and 6/90 (7%) for C. jejuni or C. coli. Except for the use of different amplification primers, this approach is identical for any bacterial species to be detected. Direct PCR analysis of food samples offers rapid screening for the presence of specific bacteria and enables selection of critical samples prior to culture.

Random amplification of polymorphic bacterial DNA: evaluation of 11 oligonucleotides and application to food contaminated with Listeria monocytogenes.

The polymerase chain reaction was used to obtain randomly-amplified polymorphic DNA (RAPD) profiles from Listeria spp. and enterobacteria. Eleven different oligonucleotides were evaluated. Only one, HR4 (19mer), generated reproducible and specific profiles for Listeria spp., while results for enterobacteria were controversial. A total of 57 different Listeria strains were subjected to the RAPD analysis and 27 different profiles were recognized. RAPD typing allowed strains of the same serotype to be distinguished but the same profile was obtained from different serotypes of L. monocytogenes in three cases and in one case two different serotypes of L. innocua yielded the same profile. RAPD-typing with HR4 allowed L. monocytogenes contamination in several food outlets to be traced back to a food processing plant. In additional experiments, the general utility of this RAPD system in typing Yersinia enterocolitica, verotoxigenic Escherichia coli and Salmonella enteritidis was evaluated.

Reliability of PCR decontamination systems.

A major problem in the application of PCR is contamination with material amplified previously. Repeated PCRs result in the accumulation of intact and degraded amplicons and primer artifacts that can contaminate following amplification reactions. Post-PCR UV treatment and pre-PCR uracil DNA glycosylase (UDG) digestion have been recognized to efficiently inactivate or decompose intact amplification fragments. We show here that degraded amplification products and primer artifacts account for decreased sensitivity and may cause false-negative results. Our experiments indicate that partly degraded PCR products and primer artifacts containing sequences homologous to the primer oligonucleotides in the succeeding PCR reaction compete efficiently with sample DNA for the primers. The experiments done in this study may explain unexpectedly low PCR sensitivities reported in an increasing number of publications. In an attempt to solve this problem, we evaluated three post-PCR treatment methods to completely eliminate sequences competing for the amplification primers, namely, 8-methoxypsoralen (MOPS) or hydroxylamine treatment of amplified DNA and use of oligonucleotides containing 5'-ChemiClamps. However, all three methods did not sufficiently inhibit artificially produced carryover contaminations. In conclusion, false-positive results can be eliminated with UDG or UV treatment, but physical barriers are indispensable to avoid the occurrence of false-negative results.

Direct detection of Listeria monocytogenes using paramagnetic bead DNA extraction and enzymatic DNA amplification.

The potential of the polymerase chain reaction (PCR) as a tool for direct detection of Listeria monocytogenes in food and clinical samples was investigated. A specific oligonucleotide, directed against the listeriolysin 0 gene of L. monocytogenes, was coupled to paramagnetic beads and used to isolate listerial DNA from food homogenates and blood. The isolated DNA was amplified with a seminested PCR procedure, which recognized the hly gene. The detection limit for L. monocytogenes in 50 ml of a buffer solution was between 1 and 10 colony forming units (cfu). In food homogenates consisting of 10 g food and 40 ml 0.85% NaCl solution, artificially spiked with an overnight culture of L. monocytogenes, the detection limit was 1-10 cfu. The method was further evaluated by application to naturally contaminated food samples. In 250 microliters whole human blood artificially spiked with L. monocytogenes 10,000 cfu could be detected.

Comparison of "Gen-Probe" DNA probe and PCR for detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese.

A comparison was made of three approaches for the detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese after enrichment. Enrichment broths were tested by plating them onto different selective agars, by "Gen Probe" DNA hybridization and by the polymerase chain reaction (PCR). Based on two-step enrichment, all three approaches showed high specificities (90% or more) in detecting L. monocytogenes. In contrast, the sensitivity of the Gen-Probe test was low (33% or less), whereas high sensitivities were obtained with selective plating and PCR (83% or more). Based on one-step enrichment, specificities again were high for selective plating and PCR assay (100%), whereas for the Gen-Probe assay the specificity was lower (88% or more). The best sensitivities were observed with selective plating (67%) and PCR (75%). In terms of sensitivity, specificity and analysis time, PCR applied to a two-step enrichment was the most powerful assay for detecting L. monocytogenes in soft and semi-soft cheese.