APPLICATION FORM Containment
To obtain approval for new organisms in containment
Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.
Application Number
APP202311
Date
www.epa.govt.nz 2
Application Form Approval for new organism in containment
Completing this application form
1. This form has been approved under section 40 of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers importing, development (production, fermentation or regeneration) or field test of any new organism (including genetically modified organisms (GMOs)) in containment. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 2. If your application is for a project approval for low-risk GMOs, please use the Containment – GMO Project application form. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application including providing advice on any consultation requirements. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise.
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Commercially sensitive information
10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available. 12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions
Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation
Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment
Any organism in which any of the genes or other genetic material: Have been modified by in vitro techniques, or Genetically Modified Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques
A new organism is an organism that is any of the following: An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998; An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation; An organism for which a containment approval has been given under the HSNO Act; New Organism An organism for which a conditional release approval has been given under the HSNO Act; A qualifying organism approved for release with controls under the HSNO Act; A genetically modified organism; An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand; An organism present in New Zealand before 29 July 1998 in contravention of
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Application Form Approval for new organism in containment
the Animals Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because: It is subject to a conditional release approval; or It is a qualifying organism approved for release with controls; or It is an incidentally imported new organism
An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal
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1. Applicant details
1.1. Applicant
Company Name: (if applicable) Life Technologies New Zealand Ltd (part of ThermoFisher Scientific)
Contact Name: Kasey Kime
Job Title: Regulatory Analyst
Physical Address: 18-24 Botha Road, Penrose, Auckland
Postal Address (provide only if not the same as the physical): PO Box 161-26, Horny, Christchurch, 8441
Phone (office and/or mobile): 03 344 4493
Fax:
Email: [email protected]
1.2. New Zealand agent or consultant (if applicable)
Company Name:
Contact Name:
Job Title:
Physical Address:
Postal Address (provide only if not the same as the physical):
Phone (office and/or mobile):
Fax:
Email:
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Application Form Approval for new organism in containment
2. Information about the application
2.1. Type of containment activity Tick the box(es) that best describe your application
Application type Type of new organism
☐ GMO Import into containment ☐x Non-GMO ☐ Develop in containment i.e. regeneration, fermentation GMO or genetic modification ☐ Non-GMO
☐ GMO Field test in containment ☐ Non-GMO
2.2. Brief application description Approximately 30 words about what you are applying to do
To import Mycoplasma fermentans into containment for use as positive control in a Mycoplasma testing assay of animal serum
2.3. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it
The Life Technologies Auckland site commercially manufactures sterile-filtered animal serum for further manufacturing applications such as the production diagnostics and vaccines. A requirement of the raw materials used in these industries is that they must be tested to the European pharmacopoeia (EP section 2.6.7) and found free of Mycoplasma. The current Mycoplasma testing performed at Life Technologies does not meet the European Pharmacopoeia. Due to this, samples of animal serum are sent away to an overseas laboratory to perform EP compliant Mycoplasma testing.
Life Technologies New Zealand plans to perform in-house Mycoplasma testing which is EP compliant. This requires the importation of a strain of Mycoplasma that is not currently recorded on the list of known organisms in New Zealand. Therefore a new application is required to obtain approval to import this organism into containment.
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Application Form Approval for new organism in containment
2.4. Background and aims of application This section is intended to put the new organism(s) in perspective of the wider activities that they will be used in. You may use more technical language but all technical words must be included in a glossary
Mycoplasma fermentans will be used as a positive control (quality control) in the Mycoplasma testing assay as specified by the European Pharmacopoeia section 2.6.7.
Order ATCC strain, rehydrate to acceptable cfu/mL
Aliquot into cryovials and freeze in liquid nitrogen
1 vial of organism would we used every fortnight as a positive control to incoculate a broth culture as part of the Mycoplasma testing assay
After 28 days of incubation the results of the quality control organism are recorded and the assay results are deemed acceptable for reporting.
The assay and controls would then be discarded into biohazardous waste.
Please find attached a summary of the Mycoplasma testing assay.
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3. Information about the new organism(s)
3.1. Name of organism Mycoplasma fermentans ATCC 19989
Mycoplasma fermentans belongs to the class Mollicutes and consist of wall-less prokaryotes which are small in size and have unusually small genomes.
The Phylum Tenericutes of the 2010 edition of Bergey’s Manual (available to download for free on the International Organization for Mycoplasmology at iom-online.org) notes Mycoplasma fermentans was first identified by Edwards in 1955. The species is described as:
“Cells are filamentous. Motility has not been established for this species. Colonies on solid media display typical fried-egg morphology. Grows well in SP-4 or Hayflick medium supplemented with either arginine or glucose at 37°C.
Pathogenicity unclear; associated with balanitis, vulvovaginitis, salpingitis, respiratory distress syndrome, pneumonia, and development of rheumatoid arthritis. Mycoplasma fermentans has also been tenuously linked with the progression of AIDS, chronic fatigue syndrome, Gulf War syndrome, Adamantiades-Behçet’s disease, and fibromyalgia.”
Mycoplasma fermentans was identified as the likely etiologic agent of an acute fatal disease in otherwise healthy adults (Lo et al, 1989a). No other infectious agents were found. A similar wasting syndrome leading to death was reported in silvered leaf monkeys after experimental infection with the same organism (Lo et al, 1989b). Many years prior to these studies, M. fermentans was isolated from bone marrow of leukemic patients (Murphy et al, 1970), and other reports associated it with rheumatoid arthritis (Bartholomew, 1967 and Williams et al, 1970). These reports prompted further investigations, including some experimental studies with animal models (Gabridge et al, 1972 and 1974 and Plata et al, 1973). Until recently, none of these studies resulted in data proving a cause- and-effect relationship between M. fermentans and human disease. A recent study by Yanez et al, 2013 successfully demonstrated an animal model of M. fermentans respiratory infection in hamsters which does seem to confirm the role of M. fermentans as a respiratory pathogen.
Mycoplasma fermentans has been tentatively associated with disease throughout its history. This is in part due to the frequently unsuccessful attempts to isolate mycoplasmas in general by routine culture methods and to the presence of individuals harbouring the organism without signs of disease. A good example of this is an early serologic study which provided evidence that antibodies to this organism are common in young adults (Taylor et al, 1966). A more recent study using polymerase chain reaction (PCR) techniques found M. fermentans DNA in 44% of saliva samples of healthy individuals (Chinghingyong and Hughes, 1996). In addition, M. fermentans DNA has been isolated from throats and urine of both healthy and non-healthy students using PCR (Ainsworth et al, 2000).
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The advent of polymerase chain reaction methodologies has enabled better detection and isolation of this fastidious organism from a wide variety of human samples. Some further examples include: the isolation of M. fermentans DNA in peripheral blood mononuclear cells of Chronic Fatigue Syndrome patients (Vojdani et al, 1998); M. fermentans DNA in the urine of samples from renal allograph recipients (Coutlee et al, 1998); M. fermentans DNA in the synovial fluid of 88% of rheumatoid arthritis patients ( Johnson et al, 2000) and 8% of patients with temporomandibular joint disorders (Watanabe et al, 1998); and M. fermentans DNA in 9.9% of genital swabs taken from HIV positive men in China (Wu et al, 2014).
Mycoplasma fermentans has also been found in genital infections of sheep (Nicholas et al, 1998).
Mycoplasma fermentans is a common contaminant of eukaryotic cell cultures. More than 20 species of Mycoplasma have been isolated from contaminated cell lines, but more than 90% of the contamination is thought to be caused by just five species of mycoplasma: Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma hyorhinis, and Mycoplasma orale, and Acholeplasma laidlawii. For this reason, M. fermentans is one of the recommended quality control organisms when performing Mycoplasma sterility testing.
Mycoplasmas are transferred by intimate contact and exchange of material between mucosal surfaces. M. fermentans may also be transmitted by deer ticks (Eskow et al, 2003).
3.2. Regulatory status of the organism
Is the organism that is the subject of this application also the subject of:
An innovative medicine application as defined in section 23A of the Medicines Act 1981?
☐ Yes ☐x No
An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?
☐ Yes ☐x No
4. Information about the containment
4.1. For field tests: The nature and method of the field test
Describe the nature and method of the field test and the experimental procedures to be used
Not applicable
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4.2. Proposed containment of the new organism(s) (physical and operational) Describe how you propose to contain the new organism(s) after taking into account its ability to escape from containment (i.e. the possible pathways for escape)
The organism will be imported into our MPI Approved Containment Facility which is approved to MAF Reg Standard 154.03.02 (Facilities for Microorganisms and Cell Cultures: 2007a). The facility is approved to operate at a minimum PC2 according to the Australian and New Zealand Standard AS/NZ Safety in Laboratories, Part 3: Microbiological aspects and containment facilities (AS/NZ 2243.3.2002).
The organism will be manipulated inside a Class II biohazardous safety cabinet by trained staff. All waste associated with the use of this organism will be discarded in the biohazardous waste.
5. Māori engagement
Discuss any engagement or consultation with Māori undertaken and summarise the outcomes. Please refer to the EPA policy ‘Engaging with Māori for applications to the EPA’ on our website (www.epa.govt.nz) or contact the EPA for advice.
As the organisms will be contained and even in the event of escape, will not present a significant risk to the interests of Māori, we did not undertake consultation with Māori. This is consistent with the EPA policy ‘Engaging with Māori for Applications to the EPA’.
6. Risks, costs and benefits
Provide information of the risks, costs and benefits of the new organism(s).
These are the positive and adverse effects referred to in the HSNO Act. It is easier to regard risks and costs as being adverse (or negative) and benefits as being positive. In considering risks, cost and benefits, it is important to look at both the likelihood of occurrence (probability) and the potential magnitude of the consequences, and to look at distribution effects (who bears the costs, benefits and risks).
Consider the adverse or positive effects in the context of this application on the environment (e.g. could the organism cause any significant displacement of any native species within its natural habitat, cause any significant deterioration of natural habitats or cause significant adverse effect to New Zealand’s inherent genetic diversity, or is the organism likely to cause disease, be parasitic, or become a vector for animal or plant disease?), human health and safety, the relationship of Māori to
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the environment, the principles of the Treaty of Waitangi, society and the community, the market economy and New Zealand’s international obligations.
You must fully complete this section referencing supporting material. You will need to provide a description of where the information in the application has been sourced from, e.g. from in-house research, independent research, technical literature, community or other consultation, and provide that information with this application.
Environmental Risks
Mycoplasma fermentans requires specific nutritional and atmospheric conditions to grow and is not likely to survive without these specialized conditions. The organism is not highly infectious and has unproven pathogenicity in healthy hosts. The risk of escape into the environment is mitigated by the containment regime and the unliklihood of survival.
Occupational Exposure Risks
There is a small risk of workers exposed to aerosols when handling this organism. This is mitigated by the operation of the facility to the principles of good laboratory practices (GLP) in accordance with AS/NZS 2243.3.2002 and the use of Class II biological safety cabinets.
Relationship to Maori Risks
We do not anticipate any potential adverse effects on the relationship to Maori or the treaty of Waitangi.
Benefits
Life Technologies New Zealand will be able to manufacture products compliant to global regulations for sale in global markets.
Life Technologies will not need to export samples for testing overseas which can cause delays for customers and add cost to the final product.
Customers will receive compliant products faster.
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7. Alternative methods and potential effects from the transfer of genetic elements This section is for developments of GMOs that take place outdoors and field tests of GMOs only
Discuss if there are alternative methods of achieving the research objective. Discuss whether there could be effects resulting from the transfer of genetic elements to other organisms in or around the site of the development or field test.
Not applicable
8. Pathway determination and rapid assessment This section is for the imports of GMOs only
Under section 42B of the HSNO Act your application may be eligible for a rapid assessment. The pathway for your application will be determined after its formal receipt, based on the data provided in this application form. If you would like your application to be considered for rapid assessment (as per the criteria below), we require you to complete this section.
8.1. Discuss whether the GMO(s) to be imported fulfil the criteria The criteria are: The host organism(s) are Category 1 or 2 host organisms as per the HSNO (Low Risk Genetic Modification) Regulations The genetic modifications are Category A or B modifications as per the HSNO (Low Risk Genetic Modification) Regulations and the modifications are not listed in the Schedule of these Regulations The minimum containment of the GMO(s) will be as per the HSNO (Low Risk Genetic Modification) Regulations (PC1 or PC2 as per AS/NZS2243.3:2002)
Not applicable
9. Other information
Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application.
None
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10. Checklist This checklist is to be completed by the applicant
Application Comments/justifications All sections of the application form completed ☐X Yes ☐ No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)
Confidential data as part of a separate, ☐ Yes ☐ No identified appendix
Supplementary optional information attached:
Copies of additional references ☐X Yes ☐ No EP Mycoplasma Test Method attached
Relevant correspondence ☐ Yes ☐X No
Administration Are you an approved EPA customer? ☐ Yes ☐X No If Yes are you an: Applicant: ☐ Agent: ☐
If you are not an approved customer, payment of fee will be by: Direct credit made to the EPA bank ☐X Yes ☐ No account (preferred method of payment) ☐X Payment to follow Date of direct credit:
Cheque for application fee enclosed ☐ Yes ☐ No ☐ Payment to follow
Electronic, signed copy of application e-mailed ☐X Yes to the EPA
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Signature of applicant or person authorised to sign on behalf of applicant
☐ I am making this application, or am authorised to sign on behalf of the applicant or applicant x organisation.
☐ I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.
Kasey Kime
3rd October 2014
Signature Date
Request for information waiver under section 59 of the HSNO Act
I request for the Authority to waive any legislative information requirements (i.e. concerning the ☐ information that has been supplied in my application) that my application does not meet (tick if applicable).
Please list below which section(s) of this form are relevant to the information waiver request:
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Appendices and referenced material (if any) and glossary (if required)
Ainsworth JG, S. Hourshid, A.D.B. Webster, C.B. Gilroy, D. Taylor-Robinson. 2000. Detection of Mycoplasma fermentans in healthy students and patients with congenital immunodeficiency Journal of Infection, Volume 40, Issue 2, March 2000, Pages 138-140
Abstract Objectives: To determine the prevalence of M. fermentans at different anatomical sites in healthy subjects and in patients with congenital immunodeficiency, and to determine whether haematogenous invasion occurs among the latter. Methods: A polymerase chain reaction (PCR) assay was used to detect M. fermentans in throat swabs and urine specimens from healthy students, and from patients with congenital immunodeficiency. Peripheral blood mononuclear cells (PBMCs) from the latter group were also tested. Results: Sixty-two students provided throat swabs, of which 11 (18%) were M. fermentans-positive; 46 provided urine specimens, of which eight (17%) were positive. Of the 45 students who provided both throat and urine specimens, 12 (27%) had M. fermentans-positive samples; four in the throat and urine, four in the throat only and four in the urine only. Nineteen of the 20 patients with congenital immunodeficiency provided throat swabs, of which one (5%) was M. fermentans-positive; 19 also provided urine specimens, of which three (16%) were positive. All of the immunodeficient patients provided a PBMC sample, but none was positive. Conclusion: M. fermentans occurred frequently at mucosal sites in a healthy population and in subjects with congenital immunodeficiency. However, such a deficiency did not lead to overt haematogenous invasion.
Bartholomew LE. 1967. Ann N Y Acad Sci. Jul 28;143(1):522-34. Characterization of mycoplasma strains and antibody studies from patients with rheumatoid arthritis.
Chinghingyong M, Hughes CV. 1996. Detection of Mycoplasma fermentans in human saliva with a polymerase chain reaction-based assay Archives of Oral Biology, Volume 41, Issue 3, March 1996, Pages 311-314
Abstract Mycoplasma fermentans and other mycoplasma species may be associated with human immunodeficiency virus infection. Little is known about the ecology of this micro-organism and its natural habitat. A polymerase chain reaction (PCR)-based assay was used to detect M. fermentans in whole saliva. The hypothesis was tested that M. fermentans is present on the mucosal surfaces of the mouth and oropharynx. Whole saliva was collected from 110 adults. The 206-bp amplification product of DNA purified from these samples was detected in ethidium bromide-stained 6% polyacrylamide gels in 49 (44.5%) samples tested. All samples were confirmed by Southern blotting with a probe based on an internal sequence of the expected amplification product. The data suggest that this organism is often found in saliva and on oropharyngeal mucosal surfaces. Saliva may play a part in its transmission between individuals. Saliva sampling may be helpful in further studies of the ecology and distribution of the micro-organism in human populations.
Coutlée F, G Saint-Louis, H Voyer, P Daloze, P Ghadirian. 1998. Mycoplasma fermentans DNA is infrequently detected in urine specimens from renal transplant recipients Molecular and Cellular Probes, Volume 12, Issue 4, August 1998, Pages 201-206.
Abstract Mycoplasma fermentans is a likely causative agent of HIV-associated nephropathy. In a pilot study, M. fermentans DNA was detected with polymerase chain reaction (PCR) in urine samples from renal allograft recipients; nine (39·1%) out of 23 renal allograft recipients (most of whom had chronic allograft rejection) and none of the 20 controls, were infected with M. fermentans. A cross-sectional study was conducted to investigate the prevalence ofM. Fermentans in urine samples from renal allograft recipients. Midstream urine samples were centrifuged at 13 000×g, purified with QIA amp and tested with PCR using RW004/RW005 and an internal control to screen for the presence of inhibitors. Of the 264 participants recruited, 263 completed the questionnaire (172 men, 92 women); 53 had chronic renal allograft rejection, 106 had chronic renal dysfunction without rejection, 69 had a normal renal allograft for more than 3 months and 35 had a renal allograft for less than 3 months. All urine samples yielded positive results for the internal control. Mycoplasma fermentans DNA was detected once in prospectively collected urine samples. The only individual infected with M. fermentans was also seropositive for HIV-1. This study demonstrates that M. fermentans can be at most sporadically detected in urine from patients living with a renal allograft but is not implicated in chronic rejection of allograft.
Edward, D.G. 1955. A suggested classification and nomenclature for organisms of the pleuropneumonia group. Int. Bull. Bacteriol. Nomencl. Taxon. 5: 85–93
Eskow E, Adelson ME, Rao RV, Mordechai E. 2003. Evidence for disseminated Mycoplasma fermentans in
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New Jersey residents with antecedent tick attachment and subsequent musculoskeletal symptoms. J Clin Rheumatol. 2003 Apr;9(2):77-87.
Abstract Mycoplasma species are one of nature's most abundant groups of microbes. These bacteria inhabit a wide diversity of insect, plant, and animal species, including humans. Certain mycoplasma species have been identified in blood-sucking arthropods, including Ixodes ticks. Frequent human exposure to this genus of ticks led us to explore the possibility of tick-mediated transmission of these bacteria. We evaluated 7 residents of central New Jersey who developed fatigue, musculoskeletal symptoms, and cognitive disturbance after tick attachment. All 7 of these patients lacked both serological evidence and erythema migrans skin lesions characteristic of Lyme disease. We were able to amplify and quantitate Mycoplasma fermentans-specific DNA from their peripheral blood lymphocytes. After antimicrobial therapy, symptoms subsided, and M. fermentans DNA could no longer be detected in their blood specimens. These findings suggest that a subset of disseminated M. fermentans infections may be a vector-mediated process in humans and should be considered in patients with puzzling musculoskeletal presentations.
Gabridge MG, Abrams GD, Murphy WH. 1972. J Infect Dis. Feb;125(2):153-60. Lethal toxicity of Mycoplasma fermentans for mice.
Gabridge MG, Gamble DD. 1974. Infect Dis.Dec;130(6):664-8. Independence of leukemoid potential and toxigenicity of Mycoplasma fermentans.
Johnson S, Sidebottom D, Bruckner F and Collins D. 2000. Identification of Mycoplasma fermentans in Synovial Fluid Samples from Arthritis Patients with Inflammatory Disease J. Clin. Microbiol. January 2000 vol. 38 no. 1 90-93.
Abstract Since 1970 Mycoplasma fermentans has been suspected of being associated with rheumatoid arthritis. However, this association has been difficult to prove, and this has been our goal. The distribution of M. fermentans was studied in the synovial fluid of patients suffering from different arthritides. Samples of synovial fluid were taken from patients with well-defined disease and a clear diagnosis. After removal of the inflammatory cells and hyaluran, they were treated with proteinase K and tested by a single or fully nested PCR with primers directed against part of the two 16S rRNA genes of M. fermentans. The product was sequenced automatically, by using an ALF Express automatic sequencer, to confirm the mycoplasma species and to identify the strain since the two genes were usually found to be polymorphic. This was also true of the type strain, strain PG18. M. fermentans was detected in 23 of 26 (88%) rheumatoid arthritis patients, and four different strains were found. It was also found in 7 of 8 (88%) of the nonrheumatoid inflammatory arthritis patient group, which consisted of one patient with reactive arthritis, one patient with pauciarticular juvenile chronic arthritis, two patients with gout, two patients with ankylosing spondylitis, and two patients with psoriatic arthritis, only one of whom was infected with M. fermentans. It was not detected in any of the 10 osteoarthritis patients. M. fermentans was therefore found to be a variable and very common organism in arthritic patients with inflammatory joint exudates and may well prove to be important in the etiology of the diseases.
Lo SC, Dawson MS, Newton PB 3rd, Sonoda MA, Shih JW, Engler WF, Wang RY, Wear DJ. 1989 (a) Am J Trop Med Hyg. Sep;41(3):364-76. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients.
Abstract We studied 6 patients from 6 different geographic areas who presented with acute flu-like illnesses. The patients developed persistent fevers, lymphadenopathy or diarrhea, pneumonia, and/or heart, liver, or adrenal failure. They died in 1-7 weeks. These patients had no serological evidence of HIV infection and could not be classified as AIDS patients according to CDC criteria. The clinical signs as well as laboratory and pathological studies of these patients suggested an active infectious process, although no etiological agent was found despite extensive infectious disease work-ups during their hospitalization. Post-mortem examinations showed histopathological lesions of fulminant necrosis involving the lymph nodes, spleen, lungs, liver, adrenal glands, heart, and/or brain. No viral inclusion cells, bacteria, fungi, or parasites could be identified in these tissues using special tissue stains. We report that immunohistochemistry using rabbit antiserum raised against VLIA, the virus-like infectious agent previously identified in patients with AIDS and shown to cause fatal systemic infection in primates, revealed VLIA antigens in these necrotizing lesions. In situ hybridization using an 35S labeled VLIA-specific DNA probe also detected VLIA genetic material in the areas of necrosis. Furthermore, virus-like particles closely resembling VLIA were identified ultrastructurally in these histopathological lesions. VLIA was associated with the systemic necrotizing lesions in these previously healthy non-AIDS patients with an acute fatal disease.
Lo SC, Wang RY, Newton PB 3rd, Yang NY, Sonoda MA, Shih JW. 1989 (b) Am J Trop Med Hyg. Apr;40(4):399-409. Fatal infection of silvered leaf monkeys with a virus-like infectious agent
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(VLIA) derived from a patient with AIDS.
Abstract Four silvered leaf monkeys, inoculated with a virus-like infectious agent (VLIA) derived from transformed NIH/3T3 cells (sb51) transfected with Kaposi's sarcoma DNA of an AIDS patient, showed wasting syndromes and died in 7-9 months. Two monkeys had a transient lymphadenopathy in earlier stages. Two moribund animals showed lymphopenia. Although 3 of the VLIA inoculated monkeys had persistent low grade fever early in the infection, the animals became afebrile in the later stages. One VLIA inoculated animal had a prominent antibody response, which occurred 7 months after VLIA inoculation. The other 3 monkeys had a transient or poor antibody response in the later stages. These 3 animals revealed periodic VLIA antigenemia during the course of the experiment. A control monkey was killed 8 months after the last VLIA inoculated monkey succumbed and showed neither an antibody response nor evidence of antigenemia. VLIA-specific DNA could be directly detected in necropsy tissues of all 4 monkeys inoculated with VLIA using the polymerase chain reaction method. VLIA infection was identified in all 4 spleens, 2 of 4 livers, 1 of 2 kidneys, and all 3 brains tested from these 4 animals, but not in the tissues from the control monkey. The necropsy examination of the 4 VLIA inoculated animals revealed no opportunistic infections, acute inflammatory lesions, malignancy or cause of death other than VLIA infection. We believe that the VLIA caused a fatal systemic infection in these monkeys.
Murphy WH, Bullis C, Dabich L, Heyn R, Zarafonetis CJ. 1970. J Natl Cancer Inst. Aug;45(2):243-51. Isolation of mycoplasma from leukemic and nonleukemic patients.
Nicholas, R.A., A. Greig, S.E. Baker, R.D. Ayling, M. Heldtander, K.E Johansson, B.M. Houshaymi and R.J. Miles. 1998. Isolation of Mycoplasma fermentans from a sheep. Vet. Rec. 142: 220–221.
Plata EJ, Abell MR, Murphy WH. 1973. J Infect Dis. Nov;128(5):588-97. Induction of leukemoid disease in mice by Mycoplasma fermentans.
Taylor-Robinson D, Shirai A, Sobĕslavský O, Chanock RM. 1966. Am J Epidemiol. Sep;84(2):301-13. Serologic response to Myocplasma pneumoniae infection. II. Significance of antibody measured by different techniques. Vojdani A, P.C Choppa, C Tagle, R Andrin, B Samimi, C.W Lapp. 1998. Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with Chronic Fatigue Syndrome. FEMS Immunology and Medical Microbiology, Volume 22, Issue 4, December 1998, Pages 355-365
Abstract Mycoplasma fermentans and other Mycoplasma species are colonizers of human mucosal surfaces and may be associated with human immunodeficiency virus infection. While many infectious agents have been described in different percentages of patients with Chronic Fatigue Syndrome (CFS), little is known about the prevalence of mycoplasmas and especially M. fermentans in CFS patients. A polymerase chain reaction (PCR)-based assay was used to detect Mycoplasma genus and M. fermentans genomes in peripheral blood mononuclear cells (PBMC) of CFS patients. Blood was collected from 100 patients with CFS and 50 control subjects. The amplified products of 717 bp of Mycoplasma genus, and 206 bp of M. fermentans were detected in DNA purified from blood samples in 52% and 34% of CFS samples, respectively. In contrast, these genomes were found in only 14% and 8% of healthy control subjects respectively (P<0.0001). All samples were confirmed by Southern blot with a specific probe based on internal sequences of the expected amplification product. Several samples, which were positive for Mycoplasma genus, were negative for M. fermentans indicating that other Mycoplasma species are involved. A quantitative PCR was developed to determine the number of M. fermentans genome copies present in 1 μg of DNA for controls and CFS patients. Mycoplasma copy numbers ranging from 130 to 880 and from 264 to 2400 were detected in controls and CFS positive subjects, respectively. An enzyme immunoassay was applied for the detection of antibodies against p29 surface lipoprotein of M. fermentans to determine the relationship between M. fermentans genome copy numbers and antibody levels. Individuals with high genome copy numbers exhibited higher IgG and IgM antibodies against M. fermentans specific peptides. Isolation of this organism by culture from clinical specimens is needed in order to demonstrate specificity of signal detected by PCR in this study.
Watanabe Tsuguo, Ken-ichiro Shibata, Tetsuya Yoshikawa, Li Dong, Akira Hasebe, Hikouji Domon, Takashi Kobayashi, Yasunori Totsuka. 1998. Detection of Mycoplasma salivarium and Mycoplasma fermentans in synovial fluids of temporomandibular joints of patients with disorders in the joints. FEMS Immunology and Medical Microbiology, Volume 22, Issue 3, November 1998, Pages 241-246.
Abstract Thirty-six synovial fluid samples of temporomandibular joints were obtained from 33 patients with pain and anterior disk displacement (closed lock) in the joints. DNAs were prepared from the samples and amplified by a PCR-based assay specific for Mycoplasma salivarium or Mycoplasma fermentans. Of the 36 samples, five (14%), three (8%), and 19 (53%) were positive for M. salivarium, M. fermentans and both, respectively.
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Williams MH, Brostoff J, Roitt IM. 1970. Lancet. Aug 8;2(7667):277-80. Possible role of Mycoplasma fermentans in pathogenesis of rheumatoid arthritis.
Wu JR, Wang B, Chen LS, Yang T, Zhou LJ, Xie YX, Xu JS, Guo HX, Huan XP. 2014. Alarming incidence of genital mycoplasmas among HIV-1-infected MSM in Jiangsu, China Eur J Clin Microbiol Infect Dis.33(2):189-95.
Abstract Males who have sex with men (MSM) are considered at high risk of blood-borne and sexually transmitted infections (STIs), mainly due to the practice of unsafe sex, often combined with drug use and needle-sharing. A cross-sectional study was designed for the detection of genital mycoplasmas during the period from March 2009 to May 2010 in Jiangsu province. This work was approved by the Research ethics Committee of Jiangsu Centers for Diseases Prevention and Control (CDC), and written consent was obtained from all participants. In total, 243 human immunodeficiency virus-1 (HIV-1)-infected MSM were screened in this study. Over half of them reported a history of sexual activity with females (65.0 %), and 26.3 % reported a history of sexually transmitted diseases (STDs) other than HIV. 44.0 % of patients were in the first 2 years of their HIV infection, and 72.4 % were still in HIV progression. Of the 243 analyzed samples, all were positive for at least one kind of mycoplasma. The infection rates of Mycoplasma genitalium, M. fermentans, M. penetrans, and M. pirum were 25.5, 9.9, 2.5, and 18.5 %, respectively. The M. genitalium infection was associated with a history of sexual activity with females, and those who had sex with females showed higher infection rates. Six M. penetrans-positive patients were still in HIV infection progression and did not receive highly active antiretroviral therapy (HAART). Men who perform this particular behavior are at higher risk of Mycoplasma infections. Further molecular and epidemiological cohort studies with larger populations are needed in order to identify the role of Mycoplasma infections in HIV-1-infected MSM.
Yáñez A1, Martínez-Ramos A, Calixto T, González-Matus FJ, Rivera-Tapia JA, Giono S, Gil C, Cedillo L. 2013. BMC Res Notes. Jan 8;6:9. Animal model of Mycoplasma fermentans respiratory infection.
Abstract Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs. RESULTS: Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage. CONCLUSIONS: Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans
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