Sensitive Method for the Quantitative
Detection of Mycoplasma Infections
Aristo Vojdani, Ph.D. and Paul C. Choppa, C.L.S.

Mycoplasma is an unusual bacterium: it does not possess a cell wall and it has the smallest genome of any bacteria that can replicate independently of host cells. Recently, Mycoplasma has received renewed attention as a pathogen in humans. However, laboratory diagnosis of Mycoplasma infections is hampered by difficulties in cultivating the organism in vitro. 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 us to quantitate the Mycoplasma load in the patient. Results of Mycoplasma quantitation are expressed as the number of copies of Mycoplasma DNA per ml of blood. This quantitative assay will assist clinicians in following the efficacy and the pharmacokinetics of their recommended treatment. Mycoplasma infection has been implicated as a cofactor in AIDS, Chronic Fatigue, Arthritis, Respiratory Infection, Urethritis and Acute Pyelonephritis.

Mycoplasma infection and human
immunodeficiency virus (HIV)

Mycoplasma incognitus (fermentans) has been described as a cofactor 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), Fibromyalgia, and Rheumatoid Arthritis.

The pathogenicity of M. incognitus in
Chronic Fatigue Syndrome (CFS) patients

During the past two years we have applied PCR-based technology to determine the prevalence of M. incognitus in blood samples of CFS patients (n=250). When specific primers for M. incognitus were used, 30% 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. incognitus genome in 30% 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.

Rheumatoid Arthritis (RA)
and Mycoplasma Infection:

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. incognitus 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).

During the past months we have applied PCR-based technology to determine the prevalence of M. incognitus in blood samples of RA patients (n=250). In 40% of patients, Mycoplasma gene sequences were detected. When specific primers for M. incognitus were used, 24% of Mycoplasma positive RA patients tested positive, indicating 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.

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 sensitivity 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 (Table 1).

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

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 (Figure 1).

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 reproducible manner. Each PCR cycle is consisted of denaturation, annealing and extension, which are performed at different temperatures.

Quantitative Competitive PCR

To meet the need for a rapid, accurate, and quantitative diagnosis of Mycoplasma incognitus infection, we have developed a quantitative competitive PCR methodology. Quantitative competitive PCR utilizes an exogenously added internal standard which is composed of neutral DNA fragments, flanked by a common target sequence with the target gene-specific primers. In this PCR, one set of primers (directed to the target gene) is used to amplify both the target gene and the neutral DNA fragment. The neutral DNA fragments compete with the target DNA for the same reagents and thus act as an internal standard. The internal standard is designed to generate a PCR product of a different size than the target gene. The quantitative competitive PCR targets two templates competing
for the same primers in the same reaction. By knowing the amount of internal standard added to the reaction, one can determine the amount of target DNA present, in this case Mycoplasma. (Figure 2).

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

Treatment of Mycoplasma-positive patients with
Doxycyclin or CIPRO

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 (200 mg/d) and CIPRO (500 mg/d) for a minimum of four weeks.

 Test requirements for the detection of Mycoplasma by PCR

• Two 10 ml yellow top tubes (ACD) of blood or synovial fluid.
  Send by overnight delivery.

 Additional Tests for Chronic Fatigue Syndrome

Our ongoing research into the molecular biology and immunology of CFS includes; 2-5A synthetase, PKR, RNase L, RNase L inhibitor, Protein kinase C, Lymphokines, Cytokines, and the determination of viral loads; HIV, HTLV, EBV, CMV, Herpes Type-6, Herpes Type-7, Coxsackie, Chlamydia, and Mycoplasmas other than M. incognitus.

CONCLUSIONS

• In a microbiology laboratory it is often difficult to cultivate Mycoplasma due to its low viability and slow growth (two weeks).
• 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 yields the highest level of sensitivity and     specificity.
• PCR test results are quantitated and expressed as the number of Mycoplasma gene     copies; this enables clinicians to follow disease progression and drug efficacy.

For information regarding these and additional tests, feel free to call us.

Reference