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SENSITIVE METHOD FOR THE QUANTITATIVE DETECTION OF MYCOPLASMA INFECTIONS AND ITS RELEVANCE TO DISEASE

 

Introduction

 

Mycoplasma are the smallest free living organisms species which are present in the normal flora and pathogenic to humans, animals and plants. Mycoplasma is an unusual bacterium in that it does not possess a cell wall and it has the smallest genome of any bacteria that can replicate independently of host cells. No other group of procaryotes has been so embroiled in controversy and in establishing a clear pathogenic niche as the mycoplasmas. Their virulence determinants are undeniably complex, and their unique biological properties likely challenge the host differently from typical bacterial pathogens. Nevertheless, previous medical research has shown mycoplasma to be associated with many diseases, such as pneumonia, urogenital complications and inflammatory arthropathies. Recently, Mycoplasma has received renewed attention as a pathogenic co-factor in humans suffering from AIDS, Chronic Fatigue, Crohn’s disease, Rheumatoid Arthritis, Respiratory Infection, Urethritis and Acute Pyelonephritis.

In n the past laboratory diagnosis of mycoplasma infections have been hampered due to the small and pleomorphic nature of mycoplasma and difficulties in cultivating the organism in vitro. This is evident from controversial early research observations where different mycoplasma strains have been isolated for similar diseases with variable frequencies. Recent availability of DNA-based technology facilitates the detection of different Mycoplasma species in blood and other body fluids with a high degree of sensitivity and specificity, and can also enable the quantitation of the Mycoplasma load in the patient. This quantitative assay will assist clinicians in following the efficacy and the pharmacokinetics of their recommended treatment.

 

Identification of Mycoplasma infections by Polymerase Chain Reaction (PCR)

Different Mycoplasma species are associated with the progression of a variety of chronic diseases. Therefore, species identification and quantitation by a rapid, and sensitive laboratory test could be useful for stratification of patients in monitoring effective therapy. Accurate diagnosis of Mycoplasma is complicated because this bacterium is one of the few major pathogens that can not be easily cultivated in vitro. Serologic tests for Mycoplasma are the mainstays of laboratory diagnosis; however, these tests lack sensivity and specificity due to a poor specific immune response of the host. In addition, the sensitivity of Mycoplasma detection by DNA probes ranges between 103 and 106 colony-forming units; this sensitivity level is not sufficient for use in a clinical laboratory (Table1)

 

Table 1: Comparative sensitivities and time needed for the identification of infectious organisms.

 

Methodology	Comparative Sensitivities		Time needed for the completion of the assay
	Number of Cells	DNA ( picograms )
PCR	1-5	10-50	1-2 days
Hybridization	50-100	250-500	2-3 days
Viral pap	100-400	500-2,000	2-3 days
Culture	>1,000	5,000	>15 days
Agglutination	>1,000,000	5,000,000	Minutes

Conclusion: As the above table shows we conclude that; PCR tests are several order of magnitude more sensitive than even tests based on direct hybridization with DNA probes.

 

The Polymerase Chain Reaction (PCR)

PCR is an in vitro method for amplifying a selected nucleic acid sequence. To target the amplification to a specific DNA segment, two primers bearing the complementary sequences that are unique to the target gene are used. These two primers hybridize to opposite strands of the target DNA, thus enabling DNA polymerase to extend the sequence between them. Each cycle produces a complementary DNA strand to the target gene. Consequently, the product of each cycle are doubled, generating an exponential increase in the overall number of copies synthesized.

Different biological substances have been used as a source for DNA isolation and subsequent PCR; these include a single human sperm, a single hair strand, stool, colonic effluents, urine, CSF and blood. In a typical PCR about 1 ng of primers are added to 1 ug of DNA template, these primers define the boundaries and the specificity of the desired replicon ( the amplified DNA segment ). To this mixture, MgCl₂, dNTPs (deoxynucleotide triphosphates), and a thermostable DNA polymerase are added. The PCR reaction is performed in a thermocycler, capable of changing its temperature quickly, precisely and in a denaturation, annealing and extension, which are performed at different temperatures.

Samples that can be used for testing:

• urogenital swabs
• amniotic fluid
• blood
• synovial fluid
• throat swabs
• biopsy material
• CSF
• seminal fluid

 

Principle of Nested PCR Test

To meet the need of an accurate and more sensitive test we have developed a nested PCR procedure. The sequences of ribosomal RNA (rRNA) gene of prokaryotes, including mycoplasma, are well conserved. The lengths and sequences of the spacer region in the rRNA operon (the region between 16S and 23S gene), however, differs from species to species. Among mycoplasmas parts of this spacer region vary and some parts are well conserved. The detection procedure utilizes the PCR process where specific primers are used to:

1. amplify the spacer region using two fluorescently tagged primers (MYC1 and MYC2) on the DNA encoding rRNA of 16S and 23S
2. perform a second (nested) PCR using two primers tagged to a different fluorescent marker (MYC3 and MYC4), which are designed on the basis of a conserved region in the spacer region, and on the 23S gene.
3. the amplified region (if any) is then applied to a bacterial sequencing system run on an automated laser fluorescent genetic analysis platform for quantitation and identification (DNA fingerprint) of the organism.

Quantitation

To meet the need of an accurate and quantitative diagnosis of Mycoplasma spp. infection, we have developed a quantitative methodology using automated state-of-the-art equipment. Quantitative PCR utilizes a known concentration of pure mycoplasma DNA from a known amount of colony forming units prior to PCR, as a positive control standard. Comparison of the amount of fluorescence from amplified DNA standards to sample DNA allows accurate calculation of original starting DNA (colony forming units per ml of whole blood (Mycoplasma load)) from sample, since the PCR amplification properties have been thoroughly calculated.

This quantitative method was designed in our laboratory to quantitate Mycoplasma levels over a large linear range, which controls the differences in amplification variability seen with diverse clinical specimens. Quantitative of Mycoplasma DNA levels in clinical specimens is particularly helpful in the evaluation of antibiotic therapy as well as patient stratification.

Species Identification

When a species is suspected after the Mycoplasma PCR screen relevant published species specific primers are used for a single round PCR. If there is still doubt as to the species the relevant amplified DNA is sequenced using Big Dye Terminal DNA Sequencing procedure with the automated PE ABI Prism fluorescent genetic analyzer.

 

CONCLUSIONS:

• In a microbiology laboratory it is often difficult to cultivate Mycoplasma due to its low viability and slow growth (two weeks) and diffferent growth requirements for different species.
• Culture of Mycoplasma species is expensive, time consuming, labor intensive and it can take up to 6 weeks before becoming positive.
• Serological methods are easier to perform and less costly. However, they are also generally non-specific, insensitive, and retrospective.
• PCR-based technology for Mycoplasma yeilds the highest level of sensitivity and specificity.
• PCR test results are quantitated and expressed as the amount of Mycoplasma DNA load; this enables clinicians to follow disease progression and drug efficacy.
• The PE ABI Prism Laser fluorescent automated platform for DNA analysis allows state of the art confirmation on species and strain.

 

Importance of measuring IgG and IgM antibodies against mycoplasma fermentans

When we measured antibodies against Mycoplasma fermentans by ELISA and compared the results to the presence of DNA in the blood, we found that only in about 62% of cases where M.fermentans DNA was positive, antibodies to M.fermentans antigens were elevated significantly. In the other 38% in which the genome was positive, IgG and IgM antibodies were not detected. This may be due to the nature of the M.fermentans cell invasion, the inhibition of phagocytosis, and the lack of immune response to this organism in these individuals. On the contrary, in about 18 % of cases, the M. fermentans genome was absent but antibodies of IgG and IgM isotype were detected in their blood. Apart from the possibility of a past infection, another possibility is that these antibodies (especially IgM) produced against human tissues in some patients with autoimmune disease may cross-react with mycoplasma antigens and give false positive results. We therefore measured antibodies against a synthesized peptide corresponding to an amino-terminal segment of a membrane protein of M.fermentans and were able to reduce the degree of cross-reactivity.

 

Gold standard for detection of mycoplasma

• The polymerase chain reaction (PCR) for detection of Mycoplasma genomes is still the gold standard.
• However, confirmation of PCR should be done by nested PCR and\or southern blot and molecular probes and sequencing in order to decrease the rate of false positivity and improve false negativity.
• Antibodies (IgG, IgM and IgA) against peptide-specific mycoplasma should be performed simultaneously.
• As no diagnostic tool is 100% accurate, we suggest that nested PCR and IgG, IgM and IgA antibodies should all be performed to gain the most accurate result.

 

Urogenital Mycoplasma and Ureaplasma DNA detection by the Polymerase Chain Reaction method

The mycoplasmas are a group of microorganisms which cause a variety of human diseases involving the respiratory and genitourinary tracts, skin, joints, and central nervous system^18,31. Mycoplasma hominis, and Ureaplasma urealyticum, colonize the lower genitourinary tract and have been associated with nonspecific vaginitis, non-gonococcal urethritis, pelvic inflammatory disease, infertility, delivery of low-birth-weight infants, habitual spontaneous abortion, and premature delivery. The reported prevalence of genitourinary colonization in unselected individuals varies from <5% to 80%. Older age and frequent sexual activity are associated with a higher frequency of colonization. The prevalence for U. urealyticum is somewhat lower in the urethra of normal adult men. In women, colonization is linked to a lower socioeconomic status, ethnicity and oral contraceptive use. U. urealyiticum may be transmitted to approximately 40% of infants born to infected mothers. Studies of human and animal inoculation, serological data, and antibiotic treatment as well as the organisms occurrence in immunodeficient patients provide evidence for a causal role for U. urealyticum in nonchlamydial, nongonococcal urethritis of males²⁰. Furthermore, U. urealyticum is an important cause of pneumonia and meningitis in very-low-birth-weight infants. Furthermore, detection of U. urealyticum in chorioamnion or amniotic fluid is associated with adverse pregnancy outcome. Neither race, socioeconomic status, nor method of contraception has been clearly shown to be an independent risk factor for M. hominis. Disease states, such as systemic lupus erythromatosus, have been shown to be a risk factor. Blanchard et al., clearly show that the PCR method of detection is faster (< 24 hrs) compared to culture (2 - 5 days) and equal if not more sensitive than culture.

Mycoplasma fermentans and Mycoplasma genitalium have recently been implicated in disease of humans. However, their ecological niche and mode of transmission have not been identified. Both organisms are apparently more difficult than other pathogenic mycoplasmas to cultivate on artificial media, thus contributing to difficulty in elucidating their pathogenic potential. Both M. fermentans and M. genitalium have been detected in the urogenital tract of adults and therefore have the potential of being sexually transmitted. In addition, M. fermentans has been detected in the placentas of two patients with AIDS and in amniotic fluid of two patients with chrioamnionitis suggesting the potential for transmission in utero and an association with chrioamnionitis. The polymerase chain reaction (PCR) technique has superceeded culture methods in the detection of these two mycoplasma in the urethra, cervix of sexually active adults and in amniotic fluid. In the study by Blanchard et al., M. genitalium was detected by PCR but not by culture in 11% of patients with non-gonococcal urethritis or cervicitis but not in amniotic fluid. M. fermentans was detected in 4 of the 232 amniotic fluid samples but not by either method in patients with urethritis or cervicitis. Other studies have shown, M. genitalium by PCR was detected in 9-20% of men with nongonococcal urethritis and 5% to 20% of women attending a clinic for sexually transmitted diseases. Furthermore, serological studies in humans and experimental studies in non-human primates suggest that M. genitalium may play a role in pelvic inflammatory disease.

 

Mycoplasma and immunodeficiency

An area of emerging mycoplasmal infections concerns immunodeficiency. Although patients with congenital or acquired disorders of antibody production are susceptible to a wide variety of microbial infections, the unique susceptibility of such patients to mycoplasmal infections is a growing concern, especially considering the number of occurrences, the types of mycoplasmas involved, and the difficulties posed in the therapeutic management of such infections. In addition, the increased use of prolonged or permanent immunosuppressive chemotherapy required for patients undergoing tissue or organ transplantation or treatment of various malignant diseases has also increased the risk for mycoplasmal infections from mycoplasmas that are part of the normal human mollicute flora to those acquired through animal contact.

The association between immunodeficiency and mycoplasmal infections was first reported in the mid 1970s in patients with primary hypogammaglobulinemia and infection with U. urealyticum, M. pneumoniae, Mycoplasma salivarium, and M. hominis that localized in joint tissue, frequently with destructive arthritis. Similar joint infections in hypogammaglobulinemic patients with these mycoplasmal species continue to be reported. Since most of these mollicutes, with the possible exception of M. pneumoniae, occur as part of the normal human flora, the origin of such joint infections is considered endogenous. Patients with hypogammaglobulinemia and other antibody deficiencies are also especially susceptible to mycoplasmal infections of the upper respiratory and urinary tracts caused most frequently by M. pneumoniae or U. urealyticum, respectively.

Mycoplasmal infections following organ transplantation and immunosuppressive chemotherapy were observed in the early 1980s, with both M. hominis and U. urealyticum reported most often. Although these infections most likely originated from the patient's normal microbial flora, a recent report of donor transmission of M. hominis to two lung allograft recipients suggests that donor tissue may be a more important factor in transplant infections than currently recognized.

While patients with antibody defects or those receiving immunosuppressive drugs appear to be the most susceptible to infections with mycoplasmas present in healthy tissues, emerging evidence indicates that contact with other mycoplasmas in the environment is an important hazard. For example, the direct isolation of a feline mycoplasma (M. felis) from the joint of a hypogammaglobulinemic patient with septic arthritis was recently reported, with suspected transmission occurring through a cat bite 6 months before the onset of arthritis. Other examples include fatal septicemia caused by M. arginini, a common animal mycoplasma, from blood and multiple tissue sites in a slaughter house employee who had advanced non-Hodgkin's lymphoma and hypogammaglobulinemia, and a septicemic infection with a canine mycoplasma (M. edwardii) in a patient with advanced AIDS.

One of the most critical aspects of mycoplasmal infections in immunodeficient patients is the frequent inability to control such infections with appropriate broad spectrum antibiotics. Although the tetracyclines and erythromycins are effective chemotherapeutic agents for many mycoplasmal infections, M. fermentans and M. hominis strains are usually resistant to erythromycin, and tetracycline-resistant strains of M.hominis and U. urealyticum have been reported from the lower urogenital tract of patients. However, these antibiotics and most other broad spectrum agents have limited mycoplasmacidal activity in vivo, and their efficacy eventually depends on an intact host immune system to eliminate the mycoplasmas. Most hypogammaglobulinemic patients lack the ability to mount a strong antibody response. Guidelines for managing such mycoplasmal infections in patients with immune defects should include immediate in vitro testing of the isolated mollicute against a wide range of antibiotics; expeditious administration of the antibiotic by the most appropriate route (intravenously, if warranted); prolonged therapy terminated only if there is no rapid clinical or microbiological response; and possibly administration of intravenous immunoglobulin. Clinical management of mycoplasmal infections in transplant patients is more difficult since immunoglobulins may enhance graft or organ rejection. In the absence of suitable mycoplasmacidal chemotherapeutic agents, vigorous and sustained chemotherapy with the most active antibiotic is the current method of choice.

 

Mycoplasma fermentans

Mycoplasma fermentans strains were first isolated from the lower genital tract of both adult men and women in the early 1950s, but their role in classic lower genital tract disease has not been established. Reports in the 1970s of M. fermentans in the joints of rheumatoid arthritis patients and in the bone marrow of children with leukemia raised expectations for its pathogenic potential; these findings have not been adequately confirmed. Sufficient evidence, however, has accumulated recently to establish an important and emerging role for M. fermentans in human respiratory and joint diseases. For example, M. fermentans has been detected by specific gene amplification techniques such as polymerase chain reaction (PCR) in the synovial fluid of patients with inflammatory arthritis, but not in the joints of patients with juvenile or reactive arthritis. In two other studies using PCR, M. fermentans was identified in the upper respiratory tract of 20% to 44% of both healthy and HIV-infected patients and was associated with acute respiratory distress syndrome in nonimmunocompromised persons.

Mycoplasma fermentans is considered to be a commensal in the human mucosal tissues and has often been found in saliva and oropharyngeal of 45% of healthy adults. Although mycoplasmas are recognized primarily as extracelluar parasites or pathogens of mucosal sufaces, recent evidence suggests that certain species may invade the host cells. The molecular and cellular bases for the invasion of M.fermentans from mucosal cells to the bloodstream and its colonization of blood remain unknown. Also, it remains unclear whether M.fermentans infection of white blood cells is transient, intermittent or persistent. It is not clear how these stages influence any disease progression. The invasion of host blood cells by M.fermentans is due to inhibition of phagocytosis by a variety of mechanisms, including antiphagocytic proteins such as proteases, phospholipases and by oxygen radicals produced by mycoplasmas. Mycoplasma fermentans is capable of fusing with T lymphocytes and change their characteristics of cytokine production.

 

Mycoplasma infection and human immunodeficiency virus (HIV)

AIDS

The role of mycoplasmas in accelerating the progression of AIDS could not have begun under more baffling and circuitous conditions. A virus-like agent that arose through transfection of NIH 3T3 cells with DNA from Kaposi sarcoma tissues of AIDS patients was later shown to be M. fermentans. The spotted history of M. fermentans in rheumatoid arthritis and leukemia and its frequent contamination of cell cultures, along with its contemporary link to AIDS, have been considerable impediments to overcome in its elevation to pathogenic status. However, careful and convincing independent studies by several laboratories have implicated M. fermentans as a cause of systemic infections and organ failure in AIDS patients. The isolation of M. fermentans from blood and urine samples of HIV-infected persons, its detection by PCR and immunohistochemistry in multiple tissue sites at various stages of AIDS, and its ability to stimulate CD4+ lymphocytes and other immunomodulatory activities implicate this Mycoplasma species as a cofactor in AIDS. Consistent with this possibility, M. fermentans has been shown to act synergistically with HIV to enhance cytopathic effects on human CD4+ lymphocytes. Coincident with these studies, a new Mycoplasma species, Mycoplasma penetrans, also has emerged as a potential cofactor in AIDS progression. Its isolation almost exclusively from the urine of HIV-infected patients, the extraordinarily high prevalence of antibodies against this mycoplasma in HIV-infected patients and not in HIV-seronegative persons, and its capacity to invade target cells and activate the immune system of HIV-infected patients at various stages of disease correlate with a synergistic role with HIV. Other mycoplasmas, including M. genitalium and M. pirum, have also been isolated from AIDS patients and implicated as potential cofactors. However, the proposed role of mycoplasmas as infectious agents and cofactors in AIDS-related disorders still remains a hypothesis without definitive proof. If cofactors of HIV are essential to the development of late stages of HIV-mediated disease, mycoplasmas possess all the prerequisite properties of the consummate helper. Their ability to establish covert or overt chronic and persistent infections with concomitant activation of the immune system, stimulation of cytokine production, and induction of oxidativestress correlate with increased HIV replication and disease progression.

Mycoplasma incognitus (fermentans) has been described as a cofacter in the progression of AIDS. Growing evidence shows that Mycoplasma is an opportunistic pathogen and possibly a cofactor in other chronic diseases as well. M.incognitus was shown to be associated with lesions in the kidneys of HIV-positive patients. For instance, M. incognitus was detected in 40% of HIV positive homosexual men, but in less than 1% of the general population. Mycoplasma infections accelerate the progression of HIV disease by stimulating the replication of HIV-1 through selective activation of CD4+T-lymphocytes. When a cell lysate of human Mycoplasma was added to cultured lymphocytes infected with HIV, significant enhancement of HIV replication was observed. These results relating to the consequences of opportunistic infection led us to investigate the prevalence and distribution of Mycoplasma in patients with Chronic Fatigue Syndrome (CFS), and Rheumatoid Arthritis and SLE.

Malignant TransformationAs early as the mid-1960s, mycoplasma-infected cell lines were associated with chromosomal aberrations, altered morphologies, and cell transformation. These abnormal oncogenic cell traits continued even after the apparent elimination of mycoplasmas, and evidence implied increased tumorigenicity of these transformed cells in animals. This issue has been revisited in studies demonstrating that longterm, persistent mycoplasmal infection of mouse embryo cells initiated a multistage cellular process that resulted in irreversible cell transformation, karyotypic alterations, and tumorigenicity in nude mice.

 

Gulf War / Chronic Fatigue Syndrome

One of the most controversial current medical issues is whether the multiple acute and chronic symptoms found in veterans of the Persian Gulf War were caused by chemical exposure, infectious agents, or psychological problems, or whether a Gulf War Syndrome exists at all. The clinical illness comprises a collection of symptoms, including chronic fatigue, joint pain, headaches, and skin rashes. One study suggests that pathogenic mycoplasmas are responsible for a large number of cases among veterans, on the basis of DNA hybridization and the responsiveness of veterans to prolonged antibiotic treatment. Even though the experimental evidence is sparse and incomplete and well-controlled and detailed studies by independent laboratories are needed, if the Gulf War Syndrome has infectious causes, mycoplasmas with their requisite biological credentials are potential candidates. Moreover, a significant improvement in clinical conditions of Mycoplasma-positive CFS patients, and soldiers with similar symptomatology that participated in the Persian Gulf War, were reported upon treatment with several cycles of Doxycyclin (200mg/d) and CIPRO (500mg/d) for a minimum of four weeks.

During the past two years we have applied PCR-based technology to determine the prevalence of M.fermentans in blood samples of CFS patients (n=650). When specific primers for M.fermentans were used, 38% of CFS patients tested positive. Mycoplasmas have been shown to share a complex relationship with the immune system. Stimulatory and suppressive effects on lymphocytes, as well as both specific and nonspecific reactions have been described. Among them are B-cell and T-cell activation and the induction of growth inhibitory cytokine secretion. Demonstration of the prevalence of the M.fermentans genome in 38% of CFS patients, in close temporal association with the dysregulation of cytokine production, may indicate an involvement of this organism as a major factor or cofactor in CFS pathogenesis.

 

Crohn's Disease

Several epidemiologic studies correlate respiratory infections with exacerbation of Crohn's disease and other chronic inflammatory bowel diseases. Acute onset gastrointestinal symptoms in patients with these diseases are accompanied by seroconversion to specific viral or M. pneumoniae antigens. As indicated earlier, mycoplasmas can elicit pleiotropic immune responses and are difficult to eliminate in patients despite appropriate antibiotic treatment. Steroid therapy to control gastrointestinal symptoms in these patients, along with the multifaceted biological properties associated with pathogenic mycoplasmas, may precipitate the onset of acute exacerbations of chronic inflammatory bowel disease.

In our laboratory we have examined the occurance of mycoplasma DNA in peripheral white blood cells of patients with Crohn’s disease and other inflammatory bowel conditions. Interestingly, Mycoplasma DNA was found to occur in close to 65% of Crohn’s disease and 58% of ulcerative colitis patients, the most prevalent mycoplasma species being M. fermentans. Furthermore, we have managed to correlate the M.fermentans DNA presence in peripheral white blood cells with its presence in bowel biopsy tissues of the same patients.Rheumatoid Arthritis and Other Human Arthritides

The occurrence of various Mycoplasma and Ureaplasma species in joint tissues of patients with rheumatoid arthritis, sexually transmitted reactive arthritis, and other human arthritides can no longer be ignored.

Many experts have suggested that RA may be caused by an infectious agent. Reports on the epidemiology of RA, and the fact that RA is about 30% genetic, are supportive evidence for an infectious etiology. Moreover, the autoimmune manifestations of RA are virtually identical to those seen in slow growing bacterial infections like chronic Mycobacterium. The arthritogenic potential of Mycoplasma in animals has led to extensive studies in determining their involvement in human RA. M.pulmonis and M.arthritidis are two species of Mycoplasma that produce arthritis in mice and rats. Whether these Mycoplasma species are capable of inducing RA in humans is currently under investigation. Schaeverbeke et. al., (J.Clin.Pathol.1996; 49: 824-828) reported the presence of M.fermentans in the joints of 21% of patients with RA (n=38), 20% of patients with spondyloarthropathy and peripheral arthritis (n=10), 20% of patients with psoriatic arthritis (n=100), and 13% of patients with unclassified arthritis (n=31).

M. genitalium has been implicated as an etiologic agent in certain human joint diseases. This clinical correlation began with the observation of a mixed infection of M. pneumoniae and M. genitalium in synovial fluid specimens of a nonimmunocompromised patient after an acute respiratory infection. A predominant role was not established for either Mycoplasma species in the initial respiratory disease or in the joint manifestations, although evidence to implicate postinfectious autoimmunity to both organisms was described. These findings prompted a PCR assay on synovial fluids from patients with various arthritic syndromes, which presented case reports on two of 13 patients with M. genitalium detected in joint fluids.

During the past months we have applied PCR-based technology to determine the prevalence of M.fermentans and M. genitalium blood samples of RA patients (n=120). In 60% of patients, Mycoplasma gene sequences were detected. When specific primers for M.fermentans and M. genitalium were used, 34% and 14% of Mycoplasma positive RA patients tested positive for these mycoplasma species respectively. This also indicates that other Mycoplasma species are involved in Arthritis.

In the initial stage of RA produced by Mycoplasmas, living bacteria may release toxic substances, such as hydrogen peroxide and other toxic cellular components, which may cause tissue damage. The destruction of host cells promotes the growth of Mycoplasma by liberating cellular material that can be used as nutrients by the microorganisms. In spite of its viability, Mycoplasma Infections in it’s chronic stationary phase can induce many new genes which can lead to production of superantigen and heat-shock proteins. Production of these harmful proteins lead to tissue destruction. The prevalence of Mycoplasma in this subset of RA patients may provide evidence for an infectious component in the pathogenesis of RA. Therefore, it is important to verify the triggering microbes so that a beneficial treatment with a prolonged course of antibiotics may be designed by the clinicians.

• The occurrence of various mycoplasma and ureaplasma species in joint tissues of patients with rheumatoid arthritis and other human arthritis can no longer be ignored.
• M. fermentans was suggested more than 20 years ago as a cause of rheumatoid arthritis (RA) on the basis of isolation from synovial fluids of a few patients. Recently, with PCR methodology, the M. fermentans genome was found in 40% of synovial biopsy specimens and in 21% of joints of patients with rheumatoid arthritis respectively. This genome was also found in 20% of patients with spondyloarthropathy and psoriatic arthritis and in 13% of patients with unclassified arthritis.
• M. fermentans was not detected in any speciments from patients with reactive arthritis, chronic juvenile arthritis, osteoarthritis or gouty arthritis.

Minocycline in rheumatoid arthritis

In two recently-published independent randomized trials, rheumatoid arthritis patients were treated with 100mg of oral minocycline twice daily or a placebo for a period of 26 weeks. In the minocycline group, more minocycline-treated patients than placebo showed greater than 75% improvement in swollen joint count, tender joint count and in clinical parameters such as serum C-reactive protein (CRP) level and erythrocyte sedimentation rate (ESR). In these studies, the intergroup differences were statistically significant for these findings and the mean changes over time revealed continual improvement in the minocycline-treated patients during the entire period of both studies.

This and other presently-available data on minocycline therapy in rheumatoid arthritis suggest that such treatment may be considered along with disease-modifying anti-rheumatic drugs such as methotrecate, sulfaxalazine, gold salts and hydroxychloroquine. However, additional clinical research is necessary to document the long-term efficacy on minocycline in the decreased progression of joint destruction. We believe that such long-term study about the efficacy of minocycline should be conducted on patients who are positive for mycoplasma and chlamydia genome (since we detect the chlamydia genome in blood and joint fluid of 20% of patients with rheumatoid arthritis) and not by random selection of arthritis patients. Such selection or comparison between mycoplasma- and chlamydia-positive patients with mycoplasma- and chlamydia-negative individuals may further increase the clinical efficacy of minocycline or doxycycline in future double-bind placebo studies. Furthermore, clinical trials into the efficacy of minocycline tretament of rheumatoid arthritis before cartilage destruction has occurred may be of more value into potentiating an antibiotic mechanism of action.

 

"the available data and proposed hypotheses that correlate mycoplasmas with disease pathogenesis range from definitive, provocative and titillating to inconclusive, confusing and heretical. Controversy seems to be a recurrent companion of mycoplasmas, yet good science and open-mindedness should overcome the legacy that has burdened them for decades."

Baseman and Tully, in Emerging Infectious Diseases, Volume 3, January-March, 1997.

 

Current Publications in the writing:

Detection of Mycoplasmas by polymerase chain reaction in the blood of rheumatoid arthritis patients. V. Paspaliaris, R. Hanner, K. Davis, and P.B. Stowell

Detection of Mycoplasmas by polymerase chain reaction in the blood of patients with inflammatory bowel disease. P. B. Stowell, R. Hanner, J. Whiting, V. Paspaliaris

Detection of Mycoplasmas by polymerase chain reaction in the blood of patients with chronic fatigue syndrome. V. Paspaliaris, R. Hanner, P. B. Stowell, K. Davis, J. Whiting.

Expression of mycoplasma fermentans monocyte/macrophage activator proteins in peripheral white blood cells of patients with SLE. V. Paspaliaris, M. Nolan, and R. Hanner

Immune-dysfunction induced in Sprague-Dawley rats by transfection of Mycoplasma fermentans M161Ag gene in peripheral white blood cells. M. Nolan, V. Paspaliaris

Detection of Coxiella burnetti in peripheral white blood cells of patients with Multiple Sclerosis by nested PCR. B. Liedtke, PB Stowell, J. Whiting, V. Paspaliaris

 

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Uphoff, C.C.; Brauer, S.; Grunicke, D.; Gignac, S.M.; MacLeod, R.A.F.; Quentmeier, H.;Steube, K.; Tummler, M.; Voges, M.; Wagner, B.; Drexier, H.G. Sensitivity and specificity of five different mycoplasma detection assays. Leukemia 1991;6:335-341.

Van Kupperveld, F.J.M., Van der logt, J.T.M., Angulo, A.F., Van Zoest, M.J., Quint, W.G.V., Niesters, H.G.M., Galama, J.M.D., and Melchers W.J.G. (1992) Appl. Environ. Microbiol. 58, 2606-2615.

Wang, R.Y.-H., Hu. Wu, W.S., Dawson, M.S., Shih, J.W.-K., Lo, S.-C., Selective detection of Mycoplasma fermentans by polymerase chain reaction and by using a nucleotide sequence within the insertion sequence-like element. Journal of Clinical Microbiology, 30:245-248, 1992.

Weisburg, W.G.; Tully, J.G.; Rose, D.L.; Petzel, J.P.; Oyaizu, H.Yang, D.; Mandelco, L.; Sechrest, J.; Lawrence, T.G.; Van Etten, J.; Manilooff, J.; Woese, C.R. A phylogenetic analysis of the mycoplasma: basis for their classification. J. Bacteriol. 1989; 6455-6467. Ss, 1968.

Wenzel, R., Hermann, R., Physical mapping of the Mycoplasma pneumoniae genome. Nucleic Acids Research 16:8323-8336, 1988.

Mycoplasma, arthritis:

Baccala, R., Smith, L,R., Vestberg M., Peterson, P.A., Cole B.C., Theofilopoulos, A.N., Mycoplasma arthritis mitogen, Vb engaged in mice, rats and humans, and requirement of HLA-Dra for presentation. Arth & Rhu, 35:434-442, 1992.

Breedveld, F.C., Dijkmans, B.A.C., and Mattie H. (1990) J. Rheumatol. 17, 43-46.

Brown T., Clark H.W., Bailey J.S., Rheumatoid Arthritis in the gorilla: a study of mycoplasma-host interaction in pathogenesis and treatment. Comp Path of Zoo Animals, Smithsonian Press, 259-266, 1980.

Burdge D.R., Reid G.D., Reeve, C.E., Robertson J.A., Stemke, G.W., Bowie, W.R., Septic arthritis due to dual infection with Mycoplasma hominis and Ureaplasma urealyticum J of Rhu, 15:366-371, 1988.

Cedillo, L.,Gil, C., Mayagoitia, G., Giono, S., Cuellar, Y., Yanez, A., Mycoplasma pneumoniae in rabbits. J of Rhu, 19:344-347,1992.

Clark, H.W.,Coker-Vann, M.R.,Bailey, J.S., Brown, T., Detection of mycoplasma antigens in immune complexes from rheumatoid arthritis synvial fliuds. Ann of Allegy, 60:394-398,1988.

Cole, B.C., Immune System: Implications for Disease Pathology. Immune system responses to antigens from these organisms could play key roles in diseases such as rheumatoid arthritis and AIDS. Mycoplasmology & Immunol Div of ASM, Vol 62,471-475,1996.

Behar, S.M., Porcelli, S.A., Mechanismsof autoimmune disease induction: the role of the immune response to microbial pathogens. Arthritis & Rheumatism, 38:458-476, 1995

Cole, B.C.; Ahmed, E.; Araneo, B.A.; Shelby, J.; Kamerath C.Weis; McCall, S.; Atkin, C.L. Immunomodulation in vivo by the mycoplasma arthritis superantigen. Man. Clin. Infec. Dis. 1993: 17 (suppl.1):S163-169.

Cole, B.C. and Cassell, G.H. (1979) Arth. Rheum. 22, 1375-1381.

Cole B.C., Griffiths, M.M., Triggering and excerbation of autoimmune arthritis by the Mycoplasma arthritis superantigen MAM. Arth & Rhu, 36:994-1002, 1993

Furr, P.M., Taylor-Robinson, D., and Webster, A.D.B. (1994) Ann. Rheum. Dis. 53, 183-187.

Jones, J.W., Pether, J.V.S., and Frost, R.W.P. (1995) J. Clin. Pathol. 48, 777-779.

Kloppenburg, M., Breedveld, F.C., Miltenburg, A.M.M., and Dijkmans, B.A.C. (1993) Clin. Exp. Rheumatol. 11, S113-S115.

Kloppenburg, M., Dijkmans, B.A.C., and Breedveld, F.C. (1995) Baill. Clin. Rheumatol. 9, 759-769.

Mardh, P.A., Nilsson, F.J., and Bjelle, A. (1973) Ann. Rheum. Dis. 32, 319-325.

McKendry, R.J.R., Is Rheumatoid Arthritis caused by an infection? Lancet, 345:1319-1320, 1995. Ford, D., The Mirobial Causes of Rheumatoid Arthritis. J. of Rhu, 18:1441-1442, 1991.

Paulus, H.E. (1995) Ann. Internal Med. 122, 147-148.

Schaeverbeke, T., Gilroy, C.B., B’eb’ear, C., Dehais, J., and Taylor-Robinson D. Mycoplasma fermentans, but not M. penetrans, detected by PCR assays in synovium from patients with rheumatoid arthritis and other rheumatic disorders. (1996) J. Clin Pathol. 49, 824-828

Schaeverbeke, T., Renaudin, H., Clerc, M., Lequen, L., Vernhes, J.P., De Barbeyrac, B., Bannwarth B., B’eb’ear, C., and Dehais, J. Systematic detection of mycoplasmas by culture and polymerase chain reaction (PCR) procedures in 209 synovial fluid samples. (1997) Br. J. Rheumatol. 36, 310-314.

Washburn, L.R., Cole, B.C., Gelman, M.I., Ward, J.R., Chronic arthritis of rabbits induced by mycoplasmas. I. Clinical, microbiologic and histologic features. Arthritis Rheum. 23:825-836, 1980.

Williams, M.H., Recovery of mycoplasma from rheumatoid synovial fluid. In: Third Pfizer Symposium on Rheumatic Diseases, pp. 161-189, Edinburgh University Press, 1968.

 

Chlamydia, arthritis:

Pando J.A., Yarboro, C., Ellaban, A., et al., Prevalence of Chlamydia trachomatis by PCR in the joint of patients with early rheumatoid arthritis. Arthritis Rheum, 38:S287,1995.

Mycoplasma, AIDS:

Bauer Fa, Wear DJ, Angritt P. Lo S-C. Mycoplasma fermentans (incognitus strain) infection in the kidneys of patients with acquired immunodeficiency syndrome and associated nephropathy: a light microscopic, immunohistochemical, and ultrastructural study. Human Pathol 1991:22:63-9.

Beecham, H.J.; Lo, S-C.; Lewis, D.E.; Corner, S.W.; Riley, K.J.; Oldfield E.C. Recovery from fulminant infection with Mycoplasma fermentans (incognitus strain) in non-immunocompromised host. Lancet 1991; 338:1014-1015.

Blanchard, A.; Montagnier. AIDS-associated mycoplasmas. Ann.Rev. Microbiol. 1994; 48:687-712.

Cole, B.C., Immune System: Implications for Disease Pathology. Immune system responses to antigens from these organisms could play key roles in diseases such as rheumatoid arthritis and AIDS. Mycoplasmology & Immunol Div of ASM, Vol 62,471-475,1996.

Hawkins, R.E.; Rickman, L.S.; Vermund, S.H.; Carl. M. Association of mycoplasma and human immunodeficiency virus infection: detection of amplified mycoplasma fermentans DNA in blood. The J. Infect. Dis. 1992: 165:581-585.

Hu, W.S., Wong, R.Y.-H., Liou, R-S., Shih, J.W.-K., Lo, S.-C., A newly recognised human pathogen Mycoplasma incognitus. Gene, 93:67-72, 1990.

Katseni, V.L.; Gilroy, C.B.; Ryait, B.J.; Ariyoshi, K.; Bienaisz, B.P.; Weber, J.N.; Robinson, T. Mycoplasma fermentans in individuals seropositive and seronegative for HIV-1. Lancet 1993; 341:271-273.

Kovacic, R.; Launay, V.; Tuppin, P.; Lafevillade, A.; Feuillie V.; Montagneir, L; Grau, O. Search for the presence pf six mycoplasma species in peripheral blood mononuclear cells of subjects seropositive and seronegative for human immunodeficiency virus. J. Clin. Microbiol. 1996; 34: 1808-1810.

Lemaitre, M., Henin, Y., Destouesse, F., ferrieux, C., Montagnier, L., Blanchard, A., Role of mycoplasma infection in the cytopathic effect induced by Human Immunodeficiency Virus Type I in infected cell lines. Infection and Immunity, 60:742-748. 1992

Lo. S-C.; Bucholz, C.L.; Wear, D.J.; Holm. R.C.; Marty A.M. Histopathology and doxycycline treatment in a previously healthy non-AIDS patient systematically infected by mycoplasma fermentans (incognitus strain). Modern Pathology 1991; 4:750-754.

Lo S-C, Dawson MS Newton PB III et al. Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients. Am J Trop Med Hyg 1989:41:364-76.

Lo S-C, Dawson MS, Wong DM, et al. Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical. In situ hybridization and ultrastructural study. Am J Trop Med Hyg 1989:41:601-6.

Lo S-C. Hayes MM. Wang RY-H Pierce PF, Kotani H. Shih JW-K. Newly dscovered mycoplasma isolated from patients infected with HIV. Lancet 1991:338:1415-8.

Lo S-C, Shih JW-K Newton PB III, et al. Virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma: Mycoplasma incognitus Am J Trop Med Hyg 1989:41:586-600.

Saillard, C.; Carle, P.; Bove, J.M.; BeBear, C.; Lo, S-C.; Shin, J.W.K.; Wamg, R.Y.H.; Rose, D.L; Tolly, T.G. Genetic and serologic relatedness between mycoplasma fermentans strains and a mycoplasma recently identified in tissues of AIDS and non-AIDS patients. Res. Virol. 1990; 141: 385-95.

 

Mycoplasma, treatment of:

Beecham, H.J.; Lo, S-C.; Lewis, D.E.; Corner, S.W.; Riley, K.J.; Oldfield E.C. Recovery from fulminant infection with Mycoplasma fermentans (incognitus strain) in non-immunocompromised host. Lancet 1991; 338:1014-1015.

Hannan, P.C.T. Observations on the possible origin of mycoplasma fermentans incognitus strain based on antobiotic sensitivity test. Antimicrob. Chemother. 1997; 39: 25-30.

Lo. S-C.; Bucholz, C.L.; Wear, D.J.; Holm. R.C.; Marty A.M. Histopathology and doxycycline treatment in a previously healthy non-AIDS patient systematically infected by mycoplasma fermentans (incognitus strain). Modern Pathology 1991; 4:750-754.

Montagnier, L., Blanchard, A., Mycoplasmas as cofactors in infection due to the Human Immunodeficiency Virus. Clinical Infectious Diseases, 17 (Suppl.1): 309-315, 1993.

Nicolson, G.L., Nicolson, N.L., Doxycycline treatment and Desert Storm. J Am Med Assoc, 273:618-619, 1995.

Mycoplasma, urogenital complications:

Berman SM, Harrison HR, Boyce WT, Haffner WJJ, Lewis M. Arthus JB: Low birth weight, prematurity and postpartum endometritis: association with prenatal cervical Mycoplasma hominis and Chlamydia trachomatis infections. JAMA 257: 1189-1194,1987.

Blanchard A, Hamrick W, Duffy L, Baldus K, Cassell GH. Use of polymerase chain reaction for detection of Mycoplasma fermentans and Mycoplasma genitalium in the urogenital tract and amniotic fluid. Clin Infect Dis 1993:17(suppl 1):S148-153.

Blanchard A Hentschel J. Duffy L.Baldus K. Cassell GH. Detection of Ureaplasma urealyticum by polymerase chain reaction in the urogenital tract of adults in amniotic fluid and I the respiratory tract of newborns. Clin Infect Dis 1993:17(suppl 1):S148-53

Braun P.Lee YH, Klein JO, Marcy SM, Klein TA, Charles DS, Levy P, Kass EH: Birth weight and genital mycoplasmas in pregnancy. N Engl J Med 284:167-171,1971.

Cassell GH, Cole BC: Mycoplasmas as agents of human disease. N Engl J Med 304:89-89, 1981

Cassell GH. Waites KB. Watson HL. Crouse DT. Harawasa R.Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns. Clin Microbiol Rev 1993:6:69-87.

Cassell GH, Waiters KB Venereal mycoplasmal infections.In:Hoeprich PD. Jordon MC. Eds. Infectious diseases. A modern treatise of infectious processes. Philadelphia: JB Lippincott. 1989:632-8.

Cassell GH. Waites KB. Crouse DT. Perinatal mycoplasmal infections Clin Perinatol 1991: 18:241-62.

Cassell GH. Waiters KB. Crouse DT. Et al. Association of Ureaplasma urealyticum infection in the lower respiratory tract with chronic lung disease and death in very-low-birth-weight infants. Lancet 1988:2:240-5.

Couch RB, Taylor-Robinson D: Mycoplasma diseases, Principles and Practise of Infectious Disease. Third edition. Edited by GL Mandell, RG Douglas, JE Bennett. New York, Churchill Livingstone, 1990.

Eschenback DA, Buchanan TM, Pollock HM: Polymicrobial etiology of acute pelvic inflammatory disease. N Engl J Med 294:166-171, 1975.

Ginsburg KS, Kundsin RB, Walter CW, Schur PH. Ureaplasma urealyticum and Mycoplasma hominis in women with systemic lupus erythromatosus. Arth Rheumat 35:429-433, 1992.

Gnarpe H, Friberg J: T mycoplasmas on spermatozoa and infertility. Nature 245:97-98,1973.

Horne HW, Kundsin RB, Kasasa TS: The role of mycoplasma infection in human reproductive failure. Fertil Steril 25:380-389, 1974.

Jensen JS Uldum SA. Sondergard-Andersen J. Vuust J.Lind K. Polymerase chain reaction for detection of Mycoplasma genitalium in clinical samples. J. Clin Microbiol 1991:29:46-50.

McCormack WM, Rosner B, Lee YH: Colonization with genital mycloplasma in women. Am J Epidemiol 97:240-245, 1973.

Sompolinsky D, Solomon F, Elkina L, Weinraub Z, Bukovsky I, Caspi E: Infections with mycoplasma and bacteria in induced midtrimester abortion and fetal loss. Am J Obstet Gynecol 121:610-616, 1975.

Stray-Pederson B, Eng J, Reiknam TM: Uterine T-mycoplasma colonization in reproductive failure. Am J Obstet Gynecol 130:207-211, 1978.

Taylor-Robinson D, McCormack WM: The genital mycoplasmas. N. Engl J Med 302:1003-1010, 1063-1067. 1980.

Taylor-Robinson D. Gilroy CB Hay PE Occurrence of Mycoplasma genitalium in different populations and its clinical significance. Clin Infect Dis 1993: 17 (suppl 1):S66-8.

Tully JG. Current status of the mollicute flora of man. Clin Infect Dis 1993:17 (suppl 1):S2-9

Waites KB. Duffy LB. Crouse DT, et al. Mycoplasmal infection of the cerebrospinal fluid in newborn infants from a community hospital population. Pediatr Infect Dis 1990:9:241-5.

Mycoplasma, Chronic Fatigue Syndrome:

Choppa, P.C.; Vojdani, A.; Tagle, C.; Andrin, R.; Magtoto, L. Multiplex PCR for the detection of Mycoplasma fermentans and M. hominis and M. penetrans in cell cultures and blood samples of patients with chronic fatigue syndrome. Mol. Cell Probes, 12:301-308, 1998.

Nicolson, G.L., Nicolson, N.L., Doxycycline treatment and Desert Storm. J Am Med Assoc, 273:618-619, 1995.

Nicolson, N.L., Nicolson G.L., The isolation, purification and analysis of specific gene-containing nucleoprotein complexes. Meth Mol Genet, 5:281-298, 1994.

Nicolson, G.L., Hyman E., Korenyi-both, A., Lopez, D.A., Nicolson, N.L., Rea, W., Urnovitz, H., Progress on Persian Gulf War illness Reality and Hypothesis. Int J Occup Med & Toxico, 4:365-370, 1995.

Vojdani, A., Mordechai, E., Flechas, J., Choppa, P., Possible Connection Between Mycoplasma Incognitus and Dysregulation of Cytokine Production in Chronic Fatigue Syndrome. Presented at the Chronic Fatigue Syndrome Research Conference, San Francisco, C.A, 1996.

Mycoplasma, lymphocytes modulation:

Dimitrov, D.S., Franzoso, G.; Salman, M.; Blumenthal, R.; Tarshis, M.; Barile, M.F.; Rottem, S.Mycoplasma fermentans (incognitus strain) cells are able to fuse with T lymphocytes. Clinical Infectious Diseases 1993; 17 (Suppl.1): S305-308.

Lemaitre, M., Henin, Y., Destouesse, F., ferrieux, C., Montagnier, L., Blanchard, A., Role of mycoplasma infection in the cytopathic effect induced by Human Immunodeficiency Virus Type I in infected cell lines. Infection and Immunity, 60:742-748. 1992

Lo S-C, Dawson MS, Wong DM, et al. Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical. In situ hybridization and ultrastructural study. Am J Trop Med Hyg 1989:41:601-6.

Marshall, A.J.; Miles, R.J.; Richards, L. The phagocytosis of mycoplasmas. J. Med. Microbiol. 1995; 43: 239-250.

Matsumoto, M.; Nishiguchi, M., Kikkawa, S.; Nishimura, H.; Nagasawa, S.; Seya, T. Structural and functional properties of complement-activating protein M161Ag, a Mycoplasma fermentans gene product that induces cytokine production by human monocytes. J. Biol. Chem. 1998 273: 12407-12414.

Mulhradt, P.F.; Schade, U. MDHM, a macrophage-stimulatory product of mycoplasma fermentans, leads to in vitro interleukin-1 (IL-1), IL-6, tumor necrosis factor and prostaglandin production and is pyrogenic in rabbits. Infec. Immun. 1991;59:3969-3974

Posnett, D.N., Hodstev, A.S., Kabak, S., Friedman, S.M.,Cole, B.C., Bhardwaj, N., Interaction of Mycoplasma arthritis superantigen with human T-cells. Clinical Infections Diseases 17:170-175, 1993.

Stuart, P.M. Mycoplasmal induction of cytokine production and major histocompatibility complex expression. Clin. Infec. Dis. 1993; 17 (Suppl.1): 5187-191.

Vojdani, A., Mordechai, E., Flechas, J., Choppa, P., Possible Connection Between Mycoplasma Incognitus and Dysregulation of Cytokine Production in Chronic Fatigue Syndrome. Presented at the Chronic Fatigue Syndrome Research Conference, San Francisco, C.A, 1996.

Mycoplasma, Crohn’s disease:

Ekbom A., Daszak P., Kraaz W., Wakefield AJ. Crohn’s disease after in-utero measles virus exposure. Lancet 348:516-517, 1996.

Mycoplasma, cell culture:

Hay, R.J.; Macy, M.L.; Chen, T. R. Mycoplasma infection of cultured cells. Nature 1989;339:487-488.

Hopert, A.; Uphoff, C.C.; Wirth, M.; Hauser, H.; Drexler, H.G. Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods of the detection of mycoplasmas contamination in cell lines. J. Immunol. Methods 1993; 164: 91-100.

McGarrity, G.J.; Kotani, M.Cell culture mycoplasmas. In: Raxin, S., Barile, M.F., eds. The Mycoplasmas. Vol. IV. New York: Academic Press 1995; 353-90.

Pawar, V.; Luczak, J.; Cox, M.S.; Dubose, J.; Harbell, J.W. Trends in the incidence and distribution of mycoplasma contamination detected in cell lines and their products. Int. Organ. Mycoplasmol. Lett. 1994;3:77.

Rawadi, G.; Dussurget, O. Advantages in PCR-based detection of mycoplasmas contaminating cell cultures. PCR Methods Appl. 1995; 4:199-208.