Aristo Vojdani is affiliated with the Immunosciences Laboratory, Inc., Beverly Hills, CA, and also with the Department of Medicine, Drew University Scholl of Medicine and Science, Los Angeles, CA.
    Al Robert Franco is affiliated with the Arthritis Center of Riverside, Riverside, CA.
    Address correspondence to: Aristo Vojdani, PhD, MT, 8730 Wilshire Boulevard, Suite 305, Beverly Hills, CA 90211 (E-mail: immunosci@ix.netcom.com).

ã 1999 by The Haworth Press, Inc. All right reserved.

KEYWORDS. Multiplex polymerase chain reaction (PCR), Mycoplasma fermentans, M. hominis, M. penetrans, and chronic fatigue syndrome, fibromyalgia, rheumatoid arthritis, and gulf war syndrome

Mycoplasmas are prokaryotes belonging to the class Mollicutes. They are the smallest free-living self- replicating bacteria known (1). Mycoplasmas have no cell wall and a very limited genome of between 600 to 1500 kbp. The fact that they have such a limited genome makes them highly dependent upon their host for survival much like viruses (1,2,11). Recent studies have implicated certain mycoplasma species as possible cofactors in a number of clinical conditions. Many different investigators have isolated Mycoplasma fermentans, M. hominis and M. penetrans from individuals suffering from a variety of diseases including chronic fatigue syndrome, rheumatoid arthritis and gulf war syndrome (3-5). Although the role that mycoplasmas may play in these clinical conditions is a subject of many debates, it does raise intrigue as to their possible involvement in chronic illnesses such as these and others with related or overlapping symptoms.

It has been well documented that people who suffer from fibromyalgia exhibit many of the same symptoms found in CFS (6,7). These two illnesses are so similar that for years many medical practitioners have considered them as the same condition. They are still regarded as closely associated in the scientific literature with only the exception of a few distinction criteria. Other research has stated the uncanny similarities between chronic fatigue syndrome and gulf war syndrome (8). Patients suffering from rheumatoid arthritis also exhibit certain symptoms characteristic to each illness (9). Although RA exhibits a narrower spectrum of clinical symptoms than CFS, FMS, and GWS it does exhibit a significant overlap of symptoms found in each condition. The fact that these four illnesses have such a high degree of similarities led us to investigate the possibility that mycoplasma infection may be a common link to each condition. A successful clinical practice greatly depends on rapid and reliable detection techniques. Mycoplasma detection by culture is laborious and may take as long as five weeks to generate results that even then may be inconclusive or inaccurate due to the fastidious nature of mycoplasmas (10-12). Enzyme-linked immunosorbent assays (ELISA) to detect antibodies directed against mycoplasma species may also be used, although it has a rapid turn-around time, the assay may lack sensitivity and specificity (13). More recently, molecular techniques using DNA probes and the polymerase chain reaction (PCR) have been developed in an attempt to improve upon current mycoplasma detection assays (14-16). Many researchers have used conventional PCR as a basis to develop multiple species detection assays called multiplex PCR (17,18). Multiplex PCR allows for the simultaneous detection and differentiation of multiple species in a single reaction with a level of sensitivity and specificity unmatched by any other diagnostic procedure (18). This assay enables clinicians to rapidly screen individuals for multiple species of potentially pathogenic mycoplasma while eliminating the cost and labor of individual assays to achieve the same results. The present study describes the use of a multiplex PCR for the detection of mycoplasmas at the genus level and the simultaneous detection and differentiation of Mycoplasma fermentans, M. hominis, and M. penetrans on clinical samples of patients with related illnesses.

A total of 100, 60, 40 and 40 CFS, RA, FMS and GWS patient samples, respectively were chosen for this study form various clinics throughout the country. The ages and the sex of the patients were matched with 160 healthy control subjects. Each patient had been ill for 1-5 years, and any other conditions that may cause CFS-like symptoms excluded individuals from the study. All blood samples were obtained under identical aseptic conditions to eliminate possible contamination and variation between samples.

The four sets of oligonucleotide primers listed in Table 1 were selected based on their ability to efficiently amplify specific target sequences under the same reaction condition.

The multiplex PCR reaction components and cycling parameters were determined through a number of experimental amplifications. We experimented with different DNA and reagent concentrations, annealing temperatures, and mycoplasma DNA copy numbers in various combinations. The optimized reaction was carried out in a final volume of 100 µl and each reaction mixture contained 10 mM TRIS-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 200 µM of each dATP, dCTP, dGTP, and dTTP, 50 DNA. The DNA amplification was performed in a GENE AMP 9600 thermal cycler (Perkin-Elmer, Norwalk, CT). The reaction parameters consisted of an initial 3 minute denaturation step at 94ºC followed by 40 amplification cycles which consisted of a denaturation step at 94ºC for 45 seconds, an annealing step at 55ºC for 1 minute, and an extension step at 72ºC for 2 minutes. The final cycle was followed by an additional extension step at 72ºC for 10 minutes a volume of 20 µl from each reaction was separated on a 1.8% agarose gel (FMC, Rockland, MD) stained with 0.5 µg ml-1 of ethidium bromide (Sigma, St. Louis, MO). The horizontal gels were submerged in TBE buffer (90 mM TRIS-borate, and 2 mM EDTA) and run at 80 V. The gels were exposed to a U.V. transilluminator to visualize the amplified products.

TABLE 1. Oligonucleotide primers used in the multiplex PCR

To determine the sensitivity level of this assay PBMCs were isolated from a healthy individual who was negative for all mycoplasma species by PCR. The cells were stained with 0.4% Trypan Blue (Sigma, St. Louis, MO) and counted using a haemocytometer. Serial dilutions of known quantities of purified mycoplasma DNA were added to 1 µg of human genomic DNA and subjected to the amplification procedure. We converted the added DNA quantities into bacterial cell copy numbers using the genome size of each mycoplasma species. This procedure enabled us to maintain control over the number of DNA templates that were added to the reaction therefore verifying the detection limit of the assay.

We also introduced various concentrations and combinations of mycoplasma DNA copies from each species into the same reaction ranging from 1 to 50 copies per µg of human genomic DNA. This was done to determine the ability of the multiplex PCR to detect multiple species in the same reaction and the level of sensitivity at which it does so. The lowest number of mycoplasma cell copies where all species were clearly visible using agarose gel electrophoresis was considered to be the detection of limit for the assay.

The reaction specificity was checked for the possibilities of cross-reactions with other mycoplasma species and closely related Gram-positive bacteria. The reaction fidelity was assessed by adding 100 ng of purified DNA from M. genitalium (49123), M. orale (23714), M. pirum (25960), M. pneumoniae (15531), M. arthritidis (35943), C. innocuum (14501), C. ramosum (25582), B. subtilis (6051), and E. coli (11775) (ATCC, Rockville, MD) to the reaction mixture in the absence and presence of negative control human genomic DNA and subjecting the samples to amplification under the conditions previously described.

Each mycoplasma species that was targeted by this assay was detectable at different bacterial cell copy numbers present among 1 µg of human genomic DNA. The Mycoplasma genus and M. fermentans primer sets had a detection limit of 7 mycoplasma DNA copies per µg of human DNA where as the M. hominis primer set was slightly less sensitive with the ability to detect 9 mycoplasma DNA copies per µg of human DNA. The M. penetrans primer set had the lowest sensitivity overall with the ability to detect 15 DNA copies of that species in the presence of 1 µg of human genomic DNA. The mycoplasma genomic DNA number detection limits were established and confirmed with the use of purified mycoplasma DNA. There was no cross-reaction with any of the species-specific primer sets when presented with control DNA from other mycoplasma species. The Mycoplasma genus primer set was able to amplify the predicted 280-bp region of each different mycoplasma species that was added to the reaction, and did not react with any of the non-mycoplasma DNA controls. Also when the multiplex PCR was applied to clinical samples the assay was able to detect each target sequence without any cross-reaction or interference from background DNA. The amplified products from actual clinical samples that consisted of M. penetrans (407 bp), M. genus (280 bp), M. fermentans (206 bp), and M. hominis (170 bp) were clearly detectable when visualized by agarose gel electrophoresis (Figure 1).

Figure 1

This multiplex PCR was used to detect the presence of mycoplasma in patients with CFS (n = 100), FMS (n = 40), RA (n = 60), and GWS (n = 60). We found that 52, 54, 49, and 55 of the patients were positive for the presence of Mycoplasma genus DNA sequences, respectively, while only 15% of healthy controls had a positive signal. Mycoplasma fermentans was detected in 32, 35, 23, and 36%, M. hominis was detected in 9, 8, 11, and 5%, and M. penetrans was detected in 6, 4, 7, and 3% of CFS, FMS, RA, and GWS patients, respectively. M. fermentans, M. hominis, and M. penetrans were detected in 8, 3, and 2% of the healthy control samples, respectively (Table 2). These results indicated that not only is mycoplasma infection occurring at a statistically significant rate in patients with CFS and related conditions over healthy controls, but also that Mycoplasma fermentans was detected at a significantly higher rate over M. hominis and M. penetrans in each sample set. M. fermentans infection averaged 32% over the combined sample sets with the highest infection rate found in GWS patients (36%) and the lowest in RA patient samples (23%). M. hominis was detected at an average of 8% over the combined sample sets with the highest infection rate found in RA patient samples (11%) and the lowest in GWS patient samples (5%). M. penetrans was detected at an average of 5% over the combined sample sets with the highest infection rate found in RA patient samples (7%) and the lowest in GWS patient samples (3%). There were mycoplasma infections detected by the genus-specific primer set that were not identified by the three species-specific primer sets used in this assay. This indicated that patients from each sample set are infected with other mycoplasma species that remain to be identified.

TABLE 2. Percentages of positive results from each sample group tested.

Mycoplasma detection has historically been a difficult and demanding task for many researchers. Recent advances in DNA technology have led to a wide variety of new detection techniques. One of the most important and profound contributions to molecular biology is the advent of the polymerase chain reaction (PCR). PCR has made possible in hours what only a short time ago would take weeks to achieve. Mycoplsama detection is no longer a difficult and frustrating task. PCR has given researchers and clinicians the ability to detect mycoplasma with a level of specificity and sensitivity unmatched by any other technique. Multiplex PCR is an extension of the practical use of PCR by which multiple DNA sequences may be detected in a single PCR reaction. It combines the rapid, sensitive, and specific character of conventional PCR with multiple species detection and differentiation in effect alleviating the cost, reagent usage, and labor of individual reactions to achieve the same result. With an increased need for rapid reliable diagnostic procedures multiplex PCR is an attractive alternative to the current detection methods available.

One application of multiple PCR is to increase the ability of clinicians to determine the causative agent in diseases with an unknown etiology such as CFS, FMS, RA, and GWS. In a recent study, our laboratory has identified mycoplasmal infection at the genus level in 52% of CFS patients and M. fermentans infection in 34% of the same sample set (3). With the use of this multiplex PCR the scope of our research has expanded to cover a more complete view of the role that mycoplasma infection may play in chronic illnesses with similar or overlapping symptoms. It is well documented that CFS, FMS, RA, and GWS share many common symptoms. Prior to this study there have been studies reporting on only the relationships of each of these illnesses individually. The present study suggests that mycoplasma infection may be the common link between these illnesses responsible in some degree for the similarities in symptoms experienced in each condition. Although mycoplasmas were found at significantly higher rate in patients suffering from these illnesses over healthy controls the role that the organisms may play in the development of symptoms remains to be determined. Using PCR based assays M. fermentans was detected in 44.5% of saliva samples collected from health adults (21). This and other studies suggested that the mouth and oropharnyx might be a significant reservoir of M. fermentans among human hosts, and that is may be a benign colonizer of the oral mucosa in the adult population (22). Although some believe M. fermentans to have a benign commensal relationship with the human host, other reports describe the pathogenic potential of M. fermentans (22-24). These reports found that M. fermentans was the only infectious agent that could be identified in biopsies or blood samples of previously health non-AIDS patients who suffered from sever chronic fatigue or flu-like illness, and that these patients recovered from this illness after adequate antimicrobial therapy (22-24). In similar studies M. fermentans DNA was detected in 5.8% of HIV infected individuals, 15% of patients with sexually transmitted diseases, and 8% of healthy controls (25). While the prevalence of this organism in HIV infection and sexually transmitted diseases were believed to be underestimated by the investigators due to the use of long-term antimicrobial treatments there does appear to be a correlation between the infection rate of healthy individuals found in that study and the present. It has yet to be determined why healthy individual are infected with M. fermentans or if they will develop symptoms characteristic of CFS and related illnesses. Further studies must be undertaken to establish the relationship that mycoplasma species have with their host, and determine the possible synergism of these organisms with other biological or even chemical agents. Still much remains unknown about the role of mycoplasmas in human disease, but with further research and the advancement of molecular techniques such as multiplex PCR we may gain more insight into the relationship between human disease and the pathogens involved.

REFERENCES