Screening for viral extraneous agents in live-attenuated avian vaccines by using a microbial microarray and sequencing.

The absence of extraneous agents (EA) in the raw material used for production and in finished products is one of the principal safety elements related to all medicinal products of biological origin, such as live-attenuated vaccines. The aim of this study was to investigate the applicability of the Lawrence Livermore Microbial detection array version 2 (LLMDAv2) combined with whole genome amplification and sequencing for screening for viral EAs in live-attenuated vaccines and specific pathogen-free (SPF) eggs. We detected positive microarray signals for avian endogenous retrovirus EAV-HP and several viruses belonging to the Alpharetrovirus genus in all analyzed vaccines and SPF eggs. We used a microarray probe mapping approach to evaluate the presence of intact retroviral genomes, which in addition to PCR analysis revealed that several of the positive microarray signals were most likely due to cross hybridization with the EAV-HPΔpol and ALV-E ev1, ev3 and ev6 loci sequences originating from the chicken genome. Sequencing of the vaccines on a MiSeq instrument verified the microarray findings and showed similar cross hybridization. Our results suggest that genomic microarrays and sequencing of avian attenuated vaccines may be applied in tests for EA.

Determination of freeze damage on HPV vaccines by use of flow cytometry.

The human papillomavirus (HPV) vaccines Gardasil, Silgard and Cervarix were labeled with antibodies against HPV strain 6 or 16/FITC conjugated secondary antibodies and analyzed by flow cytometry. The vaccines showed distinct peaks of fluorescent particles, and a shift towards decreased fluorescent particles was observed after incubation of the vaccines over night at -20 °C. Since parallel distributed vaccines could have longer route of transportation there is an increased risk of freeze damage for these types of vaccine. Shift in fluorescence of labeled vaccine particles was used to indicate whether parallel distributed Silgard, which is a vaccine type identical to Gardasil, was exposed to freeze damage during transportation, but no shift was observed. Additional experiments showed that the HPV vaccines could be degraded to smaller particles by citric acid/phosphate buffer treatment. The majority of particles detected in degraded Gardasil were very small indicating that the particles are HPV virus like particle (VLPs) labeled with antibodies, but Cervarix could only be degraded partially due to the presence of another type adjuvant in this vaccine. The described method may be useful in characterization of adjuvanted vaccines with respect to freeze damage, and to characterize vaccines containing particles corresponding to VLPs in size.

Very late relapse of PTLD 10 yr after allogeneic HSCT and nine yr after stopping immunosuppressive therapy.

We present a very late onset relapse of PTLD 10 yr after allogeneic HSCT in a patient in third remission for ALL, nine yr after the first episode of PTLD. The recipient was conditioned with fractionated TBI 12 Gy, cyclophosphamide, and horse ATG. The first episode of PTLD with a large retroperitoneal tumor occurred one yr after transplantation; a residual tumor infiltrating spleen and colon was resected one yr later. Due to continual pathological signals in liver and lungs, persistent fever, and an M-component in peripheral blood, a new course of four rituximab doses was given, after which the fever settled, the PET scan normalized, and the M-component disappeared. Without any ongoing immunosuppressive therapy, PTLD relapsed nine yr later with large intra-abdominal lymph node masses causing ureteric obstruction with bilateral hydronephrosis. Pathological features were identical to the primary PTLD tumor: EBV related, of donor origin, positive for CD138 and CD79 alpha, but negative for CD20 and CD19. The transcription factor PAX5 was negative but BOB1 and OCT2 were positive, consistent with plasmablastic lymphoma. The relapse was successfully treated with a combination of low dose chemotherapy and rituximab. Five yr after end of treatment, the girl has moderately reduced renal function but otherwise remains well without evidence of disease.

Bone marrow aspiration technique may have an impact on therapy stratification in children with acute lymphoblastic leukaemia.

BACKGROUND: Morphological evaluation of early response to chemotherapy and measurement of minimal residual disease by flow cytometry or PCR are being used for evaluation of prognosis and treatment stratification in children with acute lymphoblastic leukaemia (ALL). PROCEDURE: In a series of 14 consecutive bone marrow investigations from children with precursor B-cell ALL, morphological evaluations of smears and flow cytometric measurements of minimal residual disease in sequentially aspirated small (2 ml) and large (5-10 ml) volumes of bone marrow were compared, at various time points during therapy. RESULTS: The density of nucleated cells was markedly reduced in the large volume aspirate. The percentage of erythroblasts measured by flow cytometry was smaller, indicating dilution with peripheral cells. Similarly, the blast percentage was reduced with 54% in large aspirates, and in four instances with minimal residual disease of >0.1% in the small volume, the level of blasts in the large aspirate was below this limit. CONCLUSIONS: The amount of minimal residual disease should be measured in the first 2.5 ml of bone marrow aspirated from one puncture site. The procedure should be performed by experienced and carefully instructed doctors. In large aspirates, minimal residual disease will be underestimated, which may lead to failure to undertake a required intensification of therapy and a lower fraction of high risk patients in the trial.

Development of a primer-probe energy transfer based real-time PCR for detection of Marek's disease virus.

A real-time PCR assay, which enables simultaneous detection and differentiation of all three serotypes of Marek's disease virus, without the need for post-PCR sequencing, has been developed. The assay is based on the primer-probe energy transfer real-time PCR, which has a relatively high tolerance towards point mutations in the probe region. The PCR is followed by a probe melting point analysis, which enables confirmation of identity of amplicon and differentiation of serotypes. The assay targets the MDV031 gene, encoding UL19 major capsid protein-like protein and was shown to be quantitative, with a detection limit below 10TCID(50)/ml starting material. This sensitivity is similar to the one obtained with traditional virus cultivation. However, the PCR method can provide a laboratory result within a day, while the virus cultivation method takes more than a week to perform. The new method will be useful for testing of avian live viral vaccines and screening for extraneous agents.

Elimination of interfering activity in serum samples in the Chinese hamster ovary pertussis serology assay.

An interfering substance in various frozen serum samples was observed to inhibit the adhesion of Chinese hamster ovary (CHO) cells to microplate surfaces during a CHO pertussis neutralization test, resulting in wells that lacked cells or wells with dead cells after 2 days of incubation. The interfering activity in the serum could be eliminated by (i) transferring cells to other wells after their initial incubation, (ii) adding fetal calf serum (FCS) to the sample dilution buffer, (iii) precoating microplates with FCS, or (iv) preincubating the samples at 4 degrees C for 5 days. Preincubating the samples at 4 degrees C for 5 days reduced the interfering activity in only some of the samples. Adding serum to the sample dilution buffer or precoating the microplates with serum did not influence the antibody titers in the serum samples. The method described may be used for routine applications.

[An outbreak of measles in the county of Northern Jutland]. / Et udbrud af maeslinger i Nordjylland.

INTRODUCTION: With the introduction of MMR vaccination in the childhood vaccination programme in 1987, measles in Denmark is now rare. However, sub-optimal vaccination coverage results in the accumulation of susceptibles and outbreaks may still occur. MATERIAL AND METHODS: Information from epidemiological investigations and experience gained from the management of measles cases in a children's ward in Aalborg Hospital are described. RESULTS: A nine-year-old child was admitted with a tentative diagnosis of travel associated clinical malaria. The patient was not isolated and information on possible exposure to measles was only obtained upon discharge. Laboratory confirmation was received ten days after admission. At this time, a previous close-contact patient was re-admitted with high grade fever. This second patient was isolated following laboratory confirmation, and other hospitalised children received passive immunization. A total of 24 cases were reported, of which 12 patients were infected whilst hospitalisation and 21 were unvaccinated against measles. DISCUSSION: In a sub-optimally immunized population the introduction of measles virus may cause an outbreak in a local setting including a hospital environment, with potential spread to other geographical areas and hospitals. Obtaining a history of possible exposure to childhood infections, irrespective of the vaccination status, becomes highly significant upon admission. In the case of measles a detailed travel history is essential.