Contamination of Cell Cultures
FREEDOM OF CHOICE IS NOT FREE
Vaccination News, A Non-Profit Corporation
Picks of the Day
Archives
All the
News (includes Breaking News) - a running tab of everything posted on this website since October 29,
2003
Other archives
click
here to
download Adobe Reader
Return to: Scandals:
On "mad cows" and sick monkeys: From the people who brought you SV40 in
vaccines....
Click here for
More links re: bovine
contamination
PCR-based detection of
mycoplasma species.
Sung H,
Kang SH,
Bae YJ,
Hong JT,
Chung YB,
Lee CK,
Song S.
College of Pharmacy, Chungbuk National University, 12 Gaeshindong, Cheongju,
Chungbuk, Republic of Korea.
In this study, we describe our newly-developed sensitive two-stage PCR procedure
for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M.
bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum,
M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum).
For primary amplification, the DNA regions encompassing the 16S and 23S rRNA
genes of 13 species were targeted using general mycoplasma primers. The primary
PCR products were then subjected to secondary nested PCR, using two different
primer pair sets, designed via the multiple alignment of nucleotide sequences
obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA
fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target
DNA, and evidenced no cross-reactivity with the genomic DNA of related
microorganisms or of human cell lines, thereby confirming the sensitivity and
specificity of the primers used. The identification of contaminated species was
achieved via the performance of restriction fragment length polymorphism (RFLP)
coupled with Sau3AI digestion. The results obtained in this study furnish
evidence suggesting that the employed assay system constitutes an effective tool
for the diagnosis of mycoplasmal contamination in cell culture systems.
PMID: 16554716 [PubMed - in process]
-
Microbial contamination of cell cultures: a 2 years study.
Mirjalili A,
Parmoor E,
Moradi Bidhendi S,
Sarkari B.
Cell and Gene Bank, Biotechnology Department of Razi Vaccine and Serum Research
Institute (CGBRI), Hesarrak, Karaj, Iran. ali_mirjalili@yahoo.com
Cell line contamination is a major
drawback of main cell banks of the world and it has cost of losing important
biological products or valuable research. The causative agents are different
chemicals, invertebrates, bacteria, fungi, parasites, viral species and even
other cell lines. In this retrospective study, cell lines from various
species such as human, fish, insect, animals either offered or accessed through
usual official accession in CGBRI were studied during 2 years (2002-2004) to
detect their microbial contaminations and the causative organisms. Samples were
taken for sterility test upon cell lines receipt and upon each cell line
sub-culture. Samples were examined for bacterial (including mycoplasmas) and
fungal contamination using conventional microbiological techniques. The study
excluded parasites, viruses and other contaminating agents.
This study revealed 39% of specimens
were contaminated. The major contaminating agents were
mycoplasmas
(19%) followed by mixed infection (8%), fungi (8%) and bacteria (4%).
Among various bacterial species (except mycoplasmas) Bacillus sp., Enterococcus
sp. and Staphylococcus sp. are main bacterial agents and among various fungi
Aspergillus sp. followed by Penicillium sp., Sepedonium sp. and Botrytis sp.
were main fungal causative agents of CGBRI cell line contamination. Our study
also delineates each cell line contamination rate and its causative agents. This
is the first report of cell culture contamination from cell banks of Middle-East
countries like Iran.
PMID: 15939285 [PubMed - indexed for MEDLINE]
-
Microbiological control in stem cell banks: approaches to
standardisation.
Cobo F,
Stacey GN,
Hunt C,
Cabrera C,
Nieto A,
Montes R,
Cortes JL,
Catalina P,
Barnie A,
Concha A.
Stem Cell Bank of Andalucia (Spanish Central Node), Hospital Universitario
Virgen de las Nieves, Avda Fuerzas Armadas, 2, 18014, Granada, Spain. fernancobo@fundacionhvn.org
The transplant of cells of human origin is an increasingly complex sector of
medicine which entails great opportunities for the treatment of a range of
diseases. Stem cell banks should assure the quality, traceability and safety of
cultures for transplantation and must implement an effective programme to
prevent contamination of the final product. In donors, the presence of
infectious micro-organisms, like human immunodeficiency virus, hepatitis B
virus, hepatitis C virus and human T cell lymphotrophic virus, should be
evaluated in addition to the possibility of other new infectious agents (e.g.
transmissible spongiform encephalopathies and severe acute respiratory
syndrome). The introduction of the nucleic acid amplification can avoid the
window period of these viral infections. Contamination from the laboratory
environment can be achieved by routine screening for bacteria, fungi, yeast and
mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an
adventitious or endogenous virus, is a well-known fact that will also have to be
considered for processes involving in vitro culture of stem cells. It is also a
standard part of current good practice in stem cell banks to carry out routine
environmental microbiological monitoring of the cleanrooms where the cell
cultures and their products are prepared. The risk of viral contamination from
products of animal origin, like bovine serum and mouse fibroblasts as a "feeder
layer" for the development of embryonic cell lines, should also be considered.
Stem cell lines should be tested for prion particles and a virus of animal
origin that assure an acceptable quality.
Publication Types:
PMID: 16012832 [PubMed - indexed for MEDLINE]
-
Review: production, characterization, and testing of banked
mammalian cell substrates used to produce biological products.
Schiff LJ.
Biopharmaceutical Services, Charles River Laboratories, Ijamsville, Maryland
21754, USA. lschiff@bps.criver.com
A critical component in controlling the production of biological products
derived from human and animal cell lines is the characterization and testing of
banked cell substrates. The objective is to confirm the identity, purity, and
suitability of these cells for manufacturing use. Quality concerns for
biological products derived from cell lines originate from the presence of
cellular and adventitious contaminants. Well-characterized cell banks not only
permit a consistent source of production cells throughout the life of a product
but also decrease the likelihood of contamination by other cell lines and
adventitious agents. An important part
of qualifying a cell line is choosing the appropriate testing for the presence
of adventitious contaminants. The qualification of cell banks includes tests for
cell identity and endogenous and adventitious microbial contaminants (bacteria,
fungi, mycoplasmas,
and viruses). For cells producing recombinant deoxyribonucleic
acid-derived products, analysis of the expression construct at the nucleic acid
level (genetic stability) is also a primary concern. The strategy for designing
a safety-testing program for banked cells should be based on sound scientific
principles and current regulatory guidance.
Publication Types:
PMID: 16029074 [PubMed - indexed for MEDLINE]
Detection of
mycoplasma contaminations.
Uphoff CC,
Drexler HG.
DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany.
Mycoplasma contamination of cell lines
is one of the major problems in cell culture technology. The specific,
sensitive, and reliable detection of mycoplasma contamination is an important
part of mycoplasma control and should be an established method in every cell
culture laboratory. New cell lines as well as cell lines in continuous culture
must be tested in regular intervals. The polymerase chain reaction (PCR)
methodology offers a fast and sensitive technique to monitor all cultures in a
laboratory. The technique can also be used to determine the contaminating
mycoplasma species.The described assay can be performed within 3 h, including
sample preparation, DNA extraction, performing the PCR reaction, and analysis of
the PCR products. Special precautions necessary to avoid false-negative results
resulting from inhibitors of the Taq polymerase present in the crude samples and
the interpretation of the results are also described.
PMID: 15361652 [PubMed - indexed for MEDLINE]
-
Rapid detection and differentiation of the major
mycoplasma contaminants in cell
cultures using real-time PCR with SYBR Green I and melting curve analysis.
Harasawa R,
Mizusawa H,
Fujii M,
Yamamoto J,
Mukai H,
Uemori T,
Asada K,
Kato I.
Veterinary Microbiology, Department of Veterinary Medicine, Iwate University,
Morioka, Iwate 020-8550, Japan. harasawa-tky@umin.ac.jp
A quantitative real-time polymerase chain reaction (PCR) procedure followed by
melting curve analysis, using the green fluorescence dye SYBR Green I, was
developed for rapid detection and differentiation of mycoplasma contaminants in
cell cultures. This method showed that the detection of the target sequence was
linear over a range from 10(4) to 10 colony-forming units (CFU) of the
mycoplasma cells. Analysis of the melting temperature of the PCR products
allowed differentiation of the major mycoplasma contaminants. These results
demonstrate that the protocol described in the present study can decrease the
time to obtain reproducible results by simultaneous detection and
differentiation of the Mycoplasma species contaminating cell cultures.
Publication Types:
PMID: 16172541 [PubMed - indexed for MEDLINE]
-
Simultaneous detection and identification of common cell
culture contaminant and pathogenic mollicutes strains by reverse line blot
hybridization.
Wang H,
Kong F,
Jelfs P,
James G,
Gilbert GL.
Centre for Infectious Diseases and Microbiology Laboratory Services, Institute
of Clinical Pathology and Medical Research, Westmead, New South Wales,
Australia.
We have developed a reverse line blot (RLB)
hybridization assay to detect and identify the commonest
mollicutes
causing cell line contamination (Mycoplasma
arginini,
Mycoplasma fermentans,
Mycoplasma hyorhinis,
Mycoplasma orale,
and Acholeplasma
laidlawii)
and human infection (Mycoplasma
pneumoniae, Mycoplasma
hominis,
Mycoplasma genitalium,
Ureaplasma
parvum,
and Ureaplasma
urealyticum).
We developed a nested PCR assay with "universal" primers targeting the
mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR
products were hybridized to membrane-bound species-specific oligonucleotide
probes. The assay correctly identified reference strains of 10 mollicute
species. Cell cultures submitted for detection of mollicute contamination,
clinical specimens, and clinical isolates were initially tested by PCR assay
targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that
were positive were assessed by the RLB assay, with species-specific PCR assay as
the reference method. Initially, 100 clinical and 88 of 92 cell culture
specimens gave concordant results, including 18 in which two or more mollicute
species were detected by both methods. PCR and sequencing of the 16S-23S rRNA
intergenic spacer region and subsequent retesting by species-specific PCR assay
of the four cell culture specimens for which results were initially discrepant
confirmed the original RLB results. Sequencing of amplicons from 12 cell culture
specimens that were positive in the 16S rRNA PCR assay but negative by both the
RLB and species-specific PCR assays failed to identify any mollicute species.
The RLB hybridization assay is sensitive and specific and able to rapidly detect
and identify mollicute species from clinical and cell line specimens.
PMID: 15006769 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14634243&dopt=Abstract
-
Cell culture contamination: an overview.
Langdon SP.
Cancer Research UK Oncology Unit, Western General Hospital, Edinburgh, UK.
-
- For the cell culturist, two types
of contamination require careful monitoring and constant vigilance: the
contamination of cell cultures with microbiological organisms and the
contamination of one cell line with another. Both forms of contamination are
extremely prevalent and cannot be underestimated. Neither type can be
eliminated, only controlled and managed to minimize the possibility of
occurrence. Contamination consequences can range from minor
inconvenience (a flask of cells becoming contaminated with bacteria) to a
major disaster (published results that may be invalid owing to
cross-contamination of one cell line with another). Other types of
contaminants, such as chemical contamination, may also cause problems (e.g.,
deposits of disinfectants or detergents on glassware; residues, impurities,
and toxins in water, media or sera), but the common recurring problems are
likely to be biological in origin.
-
- Comment: Is the reason there is no mention of
possible disease transmission that it is assumed that such contamination
cannot cause disease, or has it been thoroughly tested and been shown to
(almost) never occur? (Examples
of cross-species disease transmission, and in particular that of SV40, suggest that the former
may be the case.)
PMID: 14634243 [PubMed - in process]
-
-
Social and environmental risk factors in the emergence of
infectious diseases.
Weiss RA,
McMichael AJ.
Wohl Virion Centre, Division of Infection and Immunity, University College
London, W1T 4JF, UK. rweiss@ucl.ac.uk
Fifty years ago, the age-old scourge of infectious disease was receding in the
developed world in response to improved public health measures, while the
advent of antibiotics, better vaccines, insecticides and improved surveillance
held the promise of eradicating residual problems. By the late twentieth
century, however, an increase in the emergence and re-emergence of infectious
diseases was evident in many parts of the world. This upturn looms as the
fourth major transition in human-microbe relationships since the advent of
agriculture around 10,000 years ago. About 30 new diseases have been
identified, including Legionnaires' disease, human immunodeficiency virus
(HIV)/acquired immune deficiency syndrome (AIDS), hepatitis C, bovine
spongiform encephalopathy (BSE)/variant Creutzfeldt-Jakob disease (vCJD),
Nipah virus, several viral hemorrhagic fevers and, most recently, severe acute
respiratory syndrome (SARS) and avian influenza. The emergence of these
diseases, and resurgence of old ones like tuberculosis and cholera, reflects
various changes in human ecology: rural-to-urban migration resulting in
high-density peri-urban slums; increasing long-distance mobility and trade;
the social disruption of war and conflict; changes in personal behavior; and,
increasingly, human-induced global changes, including widespread forest
clearance and climate change. Political ignorance, denial and obduracy (as
with HIV/AIDS) further compound the risks.
The use and misuse of medical
technology also pose risks, such as drug-resistant microbes and contaminated
equipment or biological medicines. A better understanding of the
evolving social dynamics of emerging infectious diseases ought to help us to
anticipate and hopefully ameliorate current and future risks.
PMID: 15577934 [PubMed - indexed for MEDLINE]
-
-
Comparison of the sensitivity of in vitro and in vivo tests
for detection of the presence of a bovine viral diarrhoea virus type 1 strain.
Antonis AF,
Bouma A,
de Bree J,
de Jong MC.
Division of Infectious Diseases and Food Chain Quality, Animal Sciences Group,
Wageningen University and Research Centre (WUR), P.O. Box 65, Lelystad AB8200,
The Netherlands. adriaan.antonis@wur.nl
Veterinary vaccines are usually tested
for the absence of contaminants. However, the quality control does not always
imply that vaccines are not contaminated as, for example, illustrated by the
bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that
contained a small amount of bovine viral diarrhoea
virus (BVDV1).
Thousands of cows were vaccinated with BHV1 vaccine
batches, and the question arose as to whether these small amounts of BVDV1, most
likely not detected with in vitro tests, could have infected cattle. More in
general, the question was whether the outcome of the in vitro tests, i.e. the in
vitro infectivity, was indicative for the infectivity for cattle, i.e. the in
vivo infectivity. We therefore carried out in vitro experiments to determine the
sensitivity of a BVDV1 isolation assay. In addition, we performed two animal
experiments, in which we estimated the lowest dose needed to infect calves with
BVDV1. We extrapolated the experimental in vitro and in vivo results from a
tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We
observed a partial response in the calves inoculated with this dose: four out of
six calves turned out to be infected. In the tissue culture test, all 20 samples
tested negative. The response in vivo, however, was not significantly higher
than the in vitro response, which implies that no difference in susceptibility
was observed between the animal test and the tissue culture test. Based on the
results in our experiments, some cattle may have been infected with BVDV1 after
the application of the contaminated BHV1 vaccine during the vaccination
campaign. The question remains that how
many cattle received contaminated vaccine, and became infected with BVDV1.
PMID: 15327789 [PubMed - indexed for MEDLINE]
-
-
Assessment of the risk of transmission of vaccine viruses by
using insufficiently cleaned injection devices.
Makoschey B,
Beer M.
Virological R & D Department, Intervet International, Wim de Korverstraat 35,
NL-5831 AN Boxmeer, The Netherlands.
PMID: 15559423 [PubMed - indexed for MEDLINE]
-
-
Rapid test for early detection of
mycoplasma contamination of the
continuous cell line J774.2.
Loudova M,
Novosad J.
Department of Clinical Immunology and Allergology, Faculty of Medicine in
Hradec Kralove, University Hospital, Charles University in Prague, Sokolska
tr. 408, 500 05 Hradec Kralove, Czech Republic. mloudova@post.cz
The authors describe a rapid, simple and inexpensive method for the routine
testing of mycoplasma contamination of the continuous mouse macrophage-like
cell line J774.2 using specific anti-mouse monoclonal antibodies (antiCD14,
antiCD80) and flow cytometry.
PMID: 15081614 [PubMed - indexed for MEDLINE]
Intersecting discourses: MMR vaccine and BSE.
Wilson C.
School of Humanities, Faculty of Arts, Education, and Social Sciences,
University of Western Sydney, Locked Bag 1797, South Penrith Distribution
Centre, NSW 1797, Australia. chris.wilson@uws.edu.au
PMID: 15971371 [PubMed - indexed for MEDLINE]
-
-
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14624801&dopt=Abstract
-
-
Detection of infectious Bovine polyomavirus.
Nairn C, Lovatt A, Galbraith DN.
Q-One Biotech Limited, Todd Campus, West of Scotland Science Park, Glasgow,
G20 0XA, UK. cnairn@q-one.co.uk
Bovine
polyomavirus
(BPyV)
is a member of the
Polyomaviridae, a virus that
was originally thought to be of simian origin but was later shown to be of
bovine origin, the primate cultures having been contaminated through the use
of foetal
bovine serum. The significance of this agent to the biotechnology industry
cannot be underestimated. The presence of
BPyV
in serum batches poses a serious risk for the contamination of human
therapeutic products. The current
PCR
based assays provide a means of detecting virus sequences but give no
indication as to the infectious nature of the virus. The communication
reports the successful development of an assay to detect infectious BPyV using
an in vitro amplification system followed by PCR. A lengthy culture period on
bovine cells was required before replicating BPyV could be detected and
distinguished from non-replicating virus in the cell culture supernatant. A
mock-test assay using foetal bovine serum positive for BPyV showed that there
was no evidence of replicating BPyV in the serum sample. The BPyV spiked serum
control showed that replicating virus was present thus confirming that the
serum itself did not inhibit replication of the virus. Cells harvested during
the culture period were subjected to fixation, embedding and sectioning and
examined by electron microscopy. Intact virus-like particles of approximately
40-50nm were observed in the nucleus of the bovine kidney cells, the site of
polyomavirus replication.
PMID: 14624801 [PubMed - in process]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12935809&dopt=Abstract
-
-
Bovine viral diarrhoea virus antigen in foetal calf serum
batches and consequences of such contamination for vaccine production.
Makoschey B, van Gelder PT, Keijsers V, Goovaerts D.
Virological R&D Department, Intervet International b.v., Wim de Korverstraat
35, NL-5831 AN, Boxmeer, The Netherlands. Birgit.Makoschey@Intervet.com
A protocol to test foetal calf serum (FCS) for contamination with bovine viral
diarrhoea virus (BVDV) is described. Following this protocol, which combines
cell culture methods and detection of pestivirus RNA,
seven batches of
FCS
were tested. Infectious BVDV
was detected in four of those batches. One of the remaining batches
contained a relatively high number of non-infectious BVDV particles. A sample
of this batch was formulated with aluminium hydroxide and aluminium phosphate
as adjuvant into an experimental vaccine preparation. This product was
injected twice into BVDV seronegative cattle with a 4 week interval. Blood
samples taken 4 weeks after the second application were negative for BVDV
specific antibodies. Our data stress
that detection of BVDV
RNA is not sufficient for a complete risk assessment on
FCS.
Discrimination between infectious and non-infectious
BVDV
is essential. This can only be achieved by cell culture methods.
PMID: 12935809 [PubMed - in process]
Detection and treatment of mycoplasma contamination in
cultured cells.
Jung H,
Wang SY,
Yang IW,
Hsueh DW,
Yang WJ,
Wang TH,
Wang HS.
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei,
ROC.
BACKGROUND: Mycoplasmas, the smallest
and simplest prokaryotes that reside in
endosomes
of mammalian cells, are widespread contaminants found in cell cultures. About
30% of all cell cultures, varying from 15 to 80%, are reportedly contaminated
with mycoplasmas.
Here, we present our experience in successfully detecting and treating
mycoplasmal infection in various cell lines. METHODS: The nested polymerase
chain reaction (PCR) detection and microscopic examination, including
phase-contrast, fluorescent, as well as differential interference contrast, were
used for detecting potential mycoplasma contamination of cell lines used in our
laboratory. As soon as mycoplasma was identified, antibiotic treatment was
initiated. RESULTS: Mycoplasmal contamination was detected in six of 15 cell
lines using the nested PCR amplification of mycoplasma DNA, which was further
demonstrated using 4, 6-Diamidino-2-phenylindole (DAPI) staining and fluorescent
microscopy. Alternate treatment with two antibiotics, macrolide (tiamulin) and
tetracycline (minocycline), effectively eliminated mycoplasma, which was
validated by both PCR and microscopic studies. CONCLUSIONS: The nested PCR using
genomic DNA extracted from cultured cells as templates is a rapid and sensitive
method for detecting mycoplasma contamination. Treatment with combined
antibiotics can completely eradicate mycoplasmal infection from cultured cells.
For the ease of use, PCR and/or DAPI staining appear suitable for detecting
potential mycoplasmal contamination in laboratories that rely heavily on the
cell culture system.
PMID: 12846524 [PubMed - indexed for MEDLINE]
-
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12421586&dopt=Abstract
-
Detection and characterization of pestivirus contaminations
in human live viral vaccines.
Studer E, Bertoni G, Candrian U.
Official Medicines Control Laboratory Biologika and R&D Unit, Division of
Biologicals, Swiss Federal Office of Public Health, P.O. Box 3003, Bern,
Switzerland.
In view of the use of potentially
contaminated foetal
calf serum (FCS)
in cell cultures pestiviruses
may be present in live viral vaccines. Thirty-six lots of human live viral
vaccines produced by three manufacturers were tested for the presence of
pestiviruses.
Bovine viral diarrhoea
virus (BVDV)
RNA was detected in 33% of the vaccine lots. All positive results were caused
by the mumps component of a single manufacturer. Partial sequences of
the 5' untranslated region of BVD viral RNA were determined. The sequences
were closely related to that of the NADL strain of BVDV. The amount of BVDV
RNA in the vaccines was determined by real-time RT-PCR using the LightCycler.
Between 3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present
in the vaccine samples. Additionally, culture tests were done with FCS and
human diploid cells used in the vaccine production of the manufacturer whose
vaccines were positive by PCR. All attempts to detect virus antigen in MRC-5
human diploid cells or to infect these cells with BVDV failed.
This suggests that
BVDV
RNA detected in human live viral vaccines represents passive carry over of
BVDV
from contaminated FCS
rather than active virus replication in human diploid cells. Our results
indicate that contamination with
BVDV
of FCS
used in vaccine production does not appear to be of immediate concern to human
health. Furthermore, our results indicate that gamma-irradiation of FCS
destroys BVDV particles and is also effective in preventing the presence of
BVDV RNA in the vaccines. Copyright 2002 The International Association for
Biologicals. Published by Elsevier Science Ltd. All rights reserved.
-
- Comment: Let's hope they are right about the
consequences re: human health..
PMID: 12421586 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11928999&dopt=Abstract
-
-
Comparative PCR analysis for detection of mycoplasma
infections in continuous cell lines.
Uphoff CC, Drexler HG.
Department of Human and Animal Cell Cultures, DSMZ-German Collection of
Microorganisms & Cell Cultures, Braunschweig. cup@dsmz.de
Mycoplasma
contamination of cell lines is one of the major problems in cell culturing.
About 15-35% of all cell lines are infected with a limited number of
mycoplasma
species of predominantly human, swine, or bovine origin. We examined
the mycoplasma contamination status in 495 cell cultures by polymerase chain
reaction (PCR) assay, microbiological culture method, and deoxyribonucleic
acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR
and DNA-RNA hybridization, in order to determine the sensitivity and
specificity of the PCR assay in routine cell culture. For those two cohorts,
results for the three or two assays were concordant in 92 and 91% of the
cases, respectively. The sensitivity (detection of true positives) of this PCR
detection assay was 86%, and the specificity (detection of true negatives) was
93%, with positive and negative predictive values (probability of correct
results) of 73 and 97%, respectively. PCR defined the mycoplasma status with
92% accuracy (detection of true positives and true negatives). The mycoplasma
contaminants were speciated by analyzing the PCR amplification fragment using
several restriction enzymes. Most of the cultures (47%) were infected with
Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M.
arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up,
PCR represents a sensitive, specific, accurate, inexpensive, and quick
mycoplasma detection assay that is suitable for the routine screening of cell
cultures.
PMID: 11928999 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11158127&dopt=Abstract
-
-
No evidence of infectious retroviruses in measles virus
vaccines produced in chicken embryo cell cultures.
Shahabuddin M, Sears JF, Khan AS.
Laboratory of Retrovirus Research, Division of Viral Products, Center for
Biologics Evaluation and Research, U.S. Food and Drug Administration,
Bethesda, Maryland 20892, USA.
All vaccines that are prepared in
chicken embryo fibroblasts (CEFs)
contain a low level of particle-associated reverse transcriptase (RT)
activity, which is produced from the avian cell substrate. The RNAs
present in the particles have sequence homology to viral DNAs belonging to the
ancient endogenous avian virus (EAV) family or to the avian sarcoma-leukosis
virus (ALV)-related subgroup E endogenous virus loci. Although no
replication-competent retrovirus has been associated with the RT activity
produced from CEFs, there have been some theoretical safety concerns regarding
potential consequences of integration of EAV and ALV sequences in human DNA,
which may result from nonproductive infection with replication-defective
particles or infection with EAV and ALV pseudotypes bearing measles virus
envelopes. To address these possibilities, we have analyzed EAV and ALV
particles in a measles virus vaccine equivalent (MVVE) preparation, obtained
from a U.S. manufacturer, for integration and for replication in human
peripheral blood mononuclear cells (PBMCs). The results show the absence of
EAV and ALV integrants in DNA prepared from MVVE-inoculated human cells by
direct DNA PCR and Alu PCR assays and no propagation of retrovirus in 18-day
cultures of MVVE-inoculated human PBMCs by a highly sensitive PCR-based RT
assay. These results provide further
confidence regarding the safety of chicken
RT
activity in live viral vaccines and support the continued use of
chick-cell-derived vaccines in humans.
Comment:
Again, let's hope they are right.
- PMID: 11158127 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11405932&dopt=Abstract
-
-
Responsibility for truth in research.
Nelson-Rees WA.
For over half a century, cell cultures
derived from animals and humans have served researchers in various fields. To
this day, cross-contamination of cultures has plagued many researchers, often
leading to mistaken results, retractions of results, cover-ups and some
out-and-out falsification of data and results following inadvertent use of the
wrong cells. Also, during years of examining cultures for purity we
learned that many virologists were not too concerned about the specificity of
the cultures they used to propagate the particular virus under study as long
as the substrate (whatever it might have been) gave optimal virus yield. Polio
virus propagates in primate cells, and much research has involved cells from
man and various species of primates. In the 1950s a large number of
chimpanzees were held in captivity in Africa for extensive studies of the
efficacy of polio vaccine in production at the Wistar Institute in
Philadelphia and elsewhere. Chimpanzee tissues, particularly kidneys, were
thus readily available and could have also provided substrates for polio virus
production, since little was known about the purity of substrates and little
attention was paid to their specificity at that time.
Publication Types:
Comment: How much concern has there been about
possible cross-species transfer of disease?
PMID: 11405932 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14609269&dopt=Abstract
-
Experimental infection of calves with bovine viral
diarrhoea virus type-2 (BVDV-2) isolated from a contaminated vaccine.
Falcone E, Cordioli P, Tarantino M, Muscillo M, Sala G, La Rosa G, Archetti
IL, Marianelli C, Lombardi G, Tollis M.
Istituto Superiore di Sanita, Laboratorio di Medicina Veterinaria, Viale
Regina Elena 299, 00161 Rome, Italy.
A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious
bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four
3-month-old calves. Severe signs of disease developed by days 4 and 6 in three
of the calves, free of BVDV and antibodies to BVDV, that had been exposed to
the virus. These calves survived the acute phase of the infection and
progressively recovered. BVDV was consistently isolated, or the respective
viral RNA was detected, in the buffy coats from blood samples collected
starting from days 2 or 4 up to days 11 or 14 after the experimental
infection. Viral RNA was also detected in sera from these infected calves
until the presence in the serum of virus neutralizing antibodies was
demonstrated. By contrast, the only calf having pre-existing neutralizing
antibodies to BVDV at the start of the study was protected from the disease.
No virus was detected at any time after experimental inoculation of this calf.
Genomic characterization of the BVDV-2 isolated in cell cultures, or detected
in sera from the experimentally infected animals, revealed 100%, homology in
the nucleotide sequence with the BVDV-2 detected as a contaminant of the live
IBR virus vaccine. These findings
provided evidence of the infective nature of the contaminant BVDV-2 and of its
potential to generate disease outbreaks when inoculated into susceptible
animals.
- Comment: Are humans susceptible?
PMID: 14609269 [PubMed - indexed for MEDLINE] -
The pathophysiology of variant Creutzfeldt-Jacob disease: the
hypotheses behind concerns for blood components and products.
Burthem J,
Roberts DJ.
Department of Biomedical Sciences, University of Manchester Institute of Science
and Technology, Manchester, UK.
Publication Types:
PMID: 12823340 [PubMed - indexed for MEDLINE]
Detection and characterization of pestivirus contaminations
in human live viral vaccines.
Studer E,
Bertoni G,
Candrian U.
Official Medicines Control Laboratory Biologika and R&D Unit, Division of
Biologicals, Swiss Federal Office of Public Health, P.O. Box 3003, Bern,
Switzerland.
In view of the use of potentially
contaminated foetal
calf serum (FCS)
in cell cultures pestiviruses
may be present in live viral vaccines. Thirty-six lots of human live viral
vaccines produced by three manufacturers were tested for the presence of pestiviruses.
Bovine viral diarrhoea
virus (BVDV)
RNA was detected in 33% of the vaccine lots. All positive results were
caused by the mumps component of a single manufacturer. Partial sequences of the
5' untranslated region of BVD viral RNA were determined. The sequences were
closely related to that of the NADL strain of BVDV. The amount of BVDV RNA in
the vaccines was determined by real-time RT-PCR using the LightCycler. Between
3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present in the
vaccine samples.Additionally, culture tests were done with FCS and human diploid
cells used in the vaccine production of the manufacturer whose vaccines were
positive by PCR. All attempts to detect virus antigen in MRC-5 human diploid
cells or to infect these cells with BVDV failed. This suggests that BVDV RNA
detected in human live viral vaccines represents passive carry over of BVDV from
contaminated FCS rather than active virus replication in human diploid cells.
Our results indicate that contamination with BVDV of FCS used in vaccine
production does not appear to be of immediate concern to human health.
Furthermore, our results indicate that gamma-irradiation of FCS destroys BVDV
particles and is also effective in preventing the presence of BVDV RNA in the
vaccines. Copyright 2002 The International Association for Biologicals.
Published by Elsevier Science Ltd. All rights reserved.
PMID: 12421586 [PubMed - indexed for MEDLINE]
Neurological adverse events associated with vaccination.
Piyasirisilp S,
Hemachudha T.
Division of Neurology, Department of Medicine, Chiang Mai University, Chiang Mai
50200, Thailand. spiyasir@mail.med.cmu.ac.th
Public tolerance to adverse reactions is minimal. Several reporting systems have
been established to monitor adverse events following immunization. The present
review summarizes data on neurologic complications following vaccination, and
provides evidence that indicates whether they were directly associated with the
vaccines. These complications include autism (measles vaccine), multiple
sclerosis (hepatitis B vaccine), meningoencephalitis (Japanese encephalitis
vaccine), Guillain-Barre syndrome and giant cell arteritis (influenza vaccine),
and reactions after exposure to animal rabies vaccine. Seizures and hypotonic/hyporesponsive
episodes following pertussis vaccination and potential risks associated with
varicella vaccination, as well as vaccine-associated paralytic poliomyelitis
following oral poliovirus vaccination, are also described.
In addition, claims that complications
are caused by adjuvants,
preservatives and contaminants [i.e. macrophagic myofasciitis
(aluminium), neurotoxicity
(thimerosal), and new variant Creutzfeldt-Jakob disease (bovine-derived
materials)] are discussed.
Publication Types:
PMID: 12045734 [PubMed - indexed for MEDLINE]
Implications of prion-induced diseases for animal-derived
pharmaceutical products.
Erstad BL.
Department of Pharmacy Practice and Science, College of Pharmacy, University of
Arizona, 1703 E. Mabel Street, Tucson, AZ 85721-0207, USA. erstad@pharmacy.arizona.edu
The implications of prion-induced diseases for the use of medications that
theoretically could harbor the infectious pathogens are discussed. Prions have
been identified as protein particles that lack nucleic acids. There is evidence
that prions cause the transmissible neurodegenerative diseases known as
transmissible spongiform encephalopathies.
Of these diseases, bovine spongiform
encephalopathy (BSE)
and the human spongiform encephalopathy to which it has been linked, new variant
Creutzfeldt-Jakob disease (CJD),
have generated the most attention. The first cases of new variant CJD
appeared in Britain in the mid-1990s. Ingestion of prion-infected beef remains
the only known cause of new variant CJD. No cases of BSE or new variant CJD have
been documented in the United States. The time from exposure to the development
of clinical sequelae appears to be about 10 years. The median duration of
illness is 14 months, and the outcome is invariably death. There is no
treatment; currently the only available approach is prevention. There is no
reliable method of predicting the number of new cases that might occur because
of lack of definitive information on the efficiency of transmission from animals
to humans and the number of people currently infected and at risk for infection.
The infectivity of medications and plasma fractionation products containing
material from cattle with BSE is unknown, but the risk is believed to be very
low. No cases of such transmission have been identified. Guidelines to keep the
risk of transmission via medications low have been promulgated by FDA, and
further research is warranted. There
have been no reports of medications or plasma fractionation products being
contaminated with the prions
that cause new variant CJD.
Ongoing vigilance and research are appropriate, however.Publication Types:
PMID: 11862637 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10650333&dopt=Abstract
-
-
Ten commandments for preventing contamination of primary
cell cultures.
Vierck JL, Byrne K, Mir PS, Dodson MV.
Department of Animal Sciences, Washington State University, Pullman,
Washington, USA.
Procedures for preventing contamination in primary cell cultures must be
carefully defined and strictly followed in order to obtain healthy cells.
Protocols have been developed and refined in our laboratory for establishing
primary cultures of muscle and fat stem cells without contamination from a
variety of animals. Contamination of cell cultures is not only frustrating,
but is also very expensive both in time and loss of materials. Through the
consistent use of proper aseptic techniques, most instances of contamination
may be avoided. We suggest that the basic principles detailed here will find
wide applicability in the culturing of primary cells without contamination
from many different types of animals and tissues.
-
- Comment: Is it merely "frustrating" and
"expensive", or are there negative health consequences associated with their
use as well?
PMID: 10650333 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10785940&dopt=Abstract
-
[Contamination of bovine fetal serum with bovine viral
diarrhea virus]
[Article in Spanish]
Zabal O, Kobrak AL, Lager IA, Schudel AA, Weber EL.
Instituto de Virologia, CICVyA, INTA Castelar, Buenos Aires, Argentina.
Fetal bovine serum (FBS) used in cell culture may be contaminated with
viruses, among them bovine viral diarrhea virus (BVDV) affecting the
production of biological reagents and the results of diagnosis. The filtration
process used in the preparation of commercial FBS abrogates most viral agents
that may be present in raw FBS, but BVDV may pass through the filters because
of its small size and its pleomorphism.
While detection of bovine herpes
virus-1 and parainfluenza-3 (PI-3) is determined by observation of the
cytopathic
effect, and also by
hemadsorption in the case of
PI-3, the most frequently isolated
BVDV
is non cytopathic,
and infects cells without morphological alterations, inducing problems that
arise after several cell generations. Batches of raw and processed FBS
were analyzed. Frequencies of BVDV detection in raw serum in Argentina were
similar to those published for USA. By conventional methods for BVDV
detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell
cultures maintained with high concentrations of the serum under study for at
least 2 weeks, with detection of viral antigen by indirect immunofluorescence,
the percentage of BVDV detection was 80%. This method shows that most lots of
commercial FBS contain BVDV. RT-PCR allows faster detection of the viral
genome, but it must be validated, as it does not show viral replication. To
eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS
is used in our laboratory.
PMID: 10785940 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10404869&dopt=Abstract
Benefits and risks due to animal serum used in cell culture
production.
Wessman SJ, Levings RL.
USDA, APHIS, VS, Center for Veterinary Biologics-Laboratory, Ames, Iowa, USA.
Infection with bovine viral
diarrhoea
virus (BVDV)
and other viruses is frequent in the bovine population. In
utero
infection leads to virus and antibody contamination of
foetal
and other serum used in cell culture production. The use of contaminated cells
for vaccine production may result in contaminated vaccines, which may lead to
seroconversion
or disease in the vaccinated animal. Contaminated serum or cell cultures may
also interfere with the diagnosis of viral infections. Methods for the
detection of BVDV and other viruses in serum, cell cultures, seed viruses and
vaccines at the CVB-L, and the frequency of detection are described. Reasons
for continued use of serum in cell culture production, and the risks of using
serum, are discussed.
PMID: 10404869 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10472327&dopt=Abstract
Cancer risk associated with simian virus 40 contaminated
polio vaccine.
Fisher SG, Weber L, Carbone M.
Cancer Cause and Prevention Program, Loyola University Medical Center,
Maywood, Illinois 60153, USA.
BACKGROUND: The presence of SV40 in monkey cell cultures used in the
preparation of the polio vaccine from 1955 through 1961 is well documented.
Investigations have consistently demonstrated the oncogenic behavior of SV40
in animal models. Early epidemiologic studies were inadequate in demonstrating
an increase in cancer incidence associated with contaminated vaccine.
Recently, investigators have provided persuasive evidence that SV40 is present
in human ependymomas, choroid plexus tumors, bone tumors, and mesotheliomas,
however, the etiologic role of the virus in tumorigenesis has not been
established. MATERIALS AND METHODS: Using data from SEER, we analyzed the
incidence of brain tumors, bone tumors, and mesotheliomas from 1973-1993 and
the possible relationship of these tumors with the administration of the SV40
contaminated vaccine. RESULTS: Our
analysis indicates increased rates of
ependymomas
(37%), osteogenic
sarcomas (26%), other bone tumors (34%) and
mesothelioma
(90%) among those in the exposed as compared to the unexposed birth cohort.
CONCLUSIONS: These data suggest that there may be an increased incidence of
certain cancers among the 98 million persons exposed to contaminated polio
vaccine in the U.S.; further investigations are clearly justified.
PMID: 10472327 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9737391&dopt=Abstract
-
Safety of biological products prepared from mammalian
cell culture. Bovine spongiform encephalopathy and other non-viral
transmissible agents.
[No authors listed]
Publication Types:
PMID: 9737391 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9542623&dopt=Abstract
-
Cell cross-contamination in cell cultures: the silent and
neglected danger.
Markovic O, Markovic N.
BioSciCon, Inc., Rockville, MD 20852, USA.
Cell cross-contamination in cell
cultures is a common problem during cell culturing and use. Contamination
invalidates research results, compromises the comparison of results between
laboratories, reduces reproducibility required in industrial production of
cell lines, and may lead to unusable therapeutic products. The problem
can be solved by increasing the awareness of its seriousness and by
introducing regular quality control of cell cross-contamination in every
laboratory where cells are grown and used.
P