What is the cause of the chronicity of Lyme Disease ?

I am from the R&D section at Microbiology Diagnostic Laboratories. Our laboratories ask the question – ‘What is the cause of the chronicity of Lyme Disease?’ Three possibilities. What if two people both getting same antibiotic treatment and both have the same bacterial load and one goes to remission and one becomes chronic – Why is this? Genetic predisposition may play a role? Also antibiotic resistance is possible? Or is it that the spirochaete itself proceeds more by the presence of a co- infection? Or also the co-infection itself?
Acknowledgments to my colleagues at MDL – where people are very knowledgeable in the field of tick-borne diseases.

We published a paper in Journal of Rheumatology :

Mycoplasma infections in patients with a history of tick bite.

Mycoplasma are very small bacteria (150 – 250 nm) and at first they were mistaken and taken to be viruses. They are prokaryotes that lack a cell wall. There are150 known species of Mycoplasma and in culture they look like fried egg colonies. The most virulent are listed here. They can be found in urino-genital tract diseases – urethritis and Mycoplasma hominis and Mycoplasma genitalium are isolated. Mycoplasma fermentans common is isolated in arathritis and has been associated with cfs and gws. In immune compromised patients one can find Mycoplasma pirium and Mycoplasma penetrans.

Paper in the Journal of Clinical Rheumatology:

Shows Disseminated M. fermentans in New Jersey resident antecedent tick attachment and subsequent muscle symptoms.

The main theme of the paper – it is the first report in which we can quantitate the amount of mycoplasma in the blood of those patient s quantitation is important and the therapy as a function of the load of the pathogen in the blood.
Paper reports on seven case reports - 4 males and 3 females, in the age range 17 to 60years. All had a tick bite history. All complain of arthralgia and fatigue symptoms.
All symptoms were similar to Lyme Disease and Babesia infection. Laboratory findings were negative for Bb (WB, PCR blood & urine, negative for all other tick borne infections as Babesia, Ehrlichia, Bartonella henselae, all negative). Found that all were positive to Mycoplasma genus PCR, quantitated amounts of M. fermentans in the blood and found variable (4 – 40/ML). Post therapy we see that , reaction to therapy, (which is for mf, Levofloxacin) all go to serum negative and symptoms improve.

I am going to tell you about two very interesting patients. Firstly patient No. 6 who was a 45 year old carpenter and he was in great shape. He had had two ticks taken from his leg. The ticks were negative for Lyme disease and TBD but positive for M. fermentans. The patient had symptoms of fatigue and intermittent fever, arthralgia, and therapy was not started straight away on opinion of physician. Over this period his bacterial load went from 14K to 16K/ml of M. fermentans. Levofloxacin therapy was started on week 3 at 550 mg/day for 10 weeks. There was a decrease in his bacterial load in week 7. At week 10, another test showed that Mycoplasma fermentans was not detected in his Blood. Twelve weeks post therapy we checked for M. fermentans and he was negative and he was in remission, and he still is now a year later.

Another patient No. 7:
Seventeen year old kid who had not been able to go to school. He had neurological involvement in this case, no concentration, fatigue, muscle pain and other symptoms. He had been an A student but he dropped to D and could not attend school on a daily basis.
We checked his Mycoplasma fermentans value which was positive and initiated therapy. However, this patient did not take the medication in a timely manner and when he attend our office two weeks later we found an increase in his bacterial load associated also with a worsening of his symptoms. Initiated Levofloxacin therapy for 2 weeks gave a great improvement, at week 6 he had resolution of symptoms and became assymptomatic he became PCR negative for Mycoplasma fermentans.

Drug of choice - Levofloxacin – a member of Quinolone family of antibiotics. These act on the bacterial enzymes ‘gyrase’ and topoisomerase. Gyrase, is a bacterial enzyme responsible for uncoiling supercoiled DNA. To replicate, bacteria must unwind their dna and this is the job of gyrase enzyme. Levofloxacin is a gyrase-inhibitor and the bacterium cannot replicate.

Our laboratory is interested in understanding antibiotic resistance – we have to understand how the enzyme works. I will not go through the chemistry. One of the findings is Levofloxacin inhibits the gyrase but when (we) did in-vitro studies to compare Levofloxacin compared to other antibiotics; we found for Mycoplasma fermentans it had a very good MIC activity, (the smaller the number the better the antibiotic), it took less antibiotic to kill more bacteria. Levofloxacin 0.016 concentration to achieve MIC50.

It’s interesting in the species of Mycoplasma - see high variety (of action) within the antibiotic killing effect. A ten-fold to a hundred-fold difference among the different antibiotics in killing the same species. It means different mechanisms for resistance within this genus.

Conclusions from this paper is that
-In a subset of patients with Mycoplasma fermentans, infection may be a vector-mediated process.
-Individuals with a range of arthritic and neurological symptoms and clinical detection of Mycoplasma fermentans responded positively to Levofloxacin treatment.
-Mycoplasma fermentans infections should be considered in patients with puzzling musculo-skeletal symptoms.

Pivotal role of PCR in the diagnosis of Co-Infection in Tick Borne Disease:

Archives of Neurology: 2001, 55. 1357 – 1363 Eskow E., Rao RVS and Mordechai E.M. (2001) Concurrent Infection of the central nervous system by Bb and Bartonella henselae.
In this paper we found another tick-borne disease - Bartonella henselae. Interesting in this study ticks were isolated from the patient.
Two males (14 and 35) and two females (15 and 30 ) all had Lyme Disease and a history of tick bite. An increase in the level of Bartonella henselae specific antibodies also detected by PCR and CSF and blood.

Also we found ticks in the household which were positive for Bartonella henselae. The vector was positive, so we had direct evidence by PCR. We found all round and indirectly by antibody testing. So this is a pathogen that can contribute to incomplete Bb resolution.

Bartonella are arbobacteria - he names four for human virulence (B. quintana, B. clarridgeiae, B. bacilliformis and B. henselae – which is associated with ‘Cat-scratch disease.
Just to make sure - ? in the tick, as differentiate from cat-scratch disease. We checked whether it was in the tick also. Lyme-like symptoms are separate from a typical cat scratch disease.

B. bacilliformis is a rare form found in South America. Bartonella quintana – associated with Trench fever prevalent in WW1 and WW2. Also in alcoholics and drug users, B. quintana is found at increased levels in those populations.

Slides of ticks:

Tick data that we have:
We have an entomologist who collects them for us – we check the ticks to see what they have in the wild.
From 123 ticks that were isolated and examined from New Jersey during February to August 2001. These were mostly Ixodes scapularis, whereas in California you get Ixodes pacificus and in Europe we have Ixodes ricinus.

What we did, we took the tick and crushed it in a buffer solution to extract the DNA from the ticks and perform PCR.
What kind of pathogens do they have? We test for Borrelia burgdorferi, Bartonella hensliae, Babesia microti, Erlichia and Mycoplasma.
We visualise quantitate the resultant electrophoresis gels and also stain and confirm results by dna sequence.

I am not an entomologist and some were not Ixodes species. The big ticks are often ‘dog ticks’ (dermacentor variabilis) and it is hard to tell the difference, so we genetically had a PCR test that can differentiate between the ticks.

Ixodes Scapularis:

We can identify molecularly. Way we do it is, we take dd16S ribosomal rna, that’s part of the genome of the tick, all the sequences of scapularis, pacificus and ricinus and in (red) areas they look alike and blue areas they are variable. So we designed specific primers to identify which tick we are interested in.

Verification:

Are the primers working? We do control also and specific primers recognise Ixodes in lanes 1,2 and 6 and do not recognise dog ticks in lane 3/4 so we can differentiate the ticks. Of the 123 ticks, we excluded 16 ticks that were dog ticks. So we have an assay that can differentiate this.
Then the data are normalised against the Ixodes scapularis tick, this is a Bartonella hensliae positive control, negative control, Bartonella is in 4/5/10 and 12.
For Mycoplasma 1,3,10 etc positive on gels for Mycoplasma
(Bar chart shows results). 43 ticks had nothing, 32 had Borrelia burgdorferi, 9 had Babesia microti, 36 - Bartonella henselae, 2 – Ehrlichia and 16 had Mycoplasma.

When we look at Co infection cases in the ticks - nine had Borrelia and Bartonella, and five had Bartonella and Mycoplasma, 4 had Borrelia and Mycoplasma. Two superstar ticks had all these infections present. (Borrelia, Bartonella, Mycoplasma and Babesia) and 2 had Borrelia and Babesia. This is very important.

An interesting aspect of co-infections that Dr Meer has been talking about - ticks not only had Lyme disease in them. Ticks are a cesspool, they have more than one pathogen in them, so you are not only looking for one pathogen in this testing.

And we have single tick co-infection cases also detected by PCR. One has Borrelia and Ehrlichia, one has Borrelia, Bartonella and Babesia and another has Baratonella, Babesia and Ehrlichia. So these ticks can occur also.

Interesting aspect also – we plotted the data of ticks collected - prevalence of pathogens in ticks.
Statistically significant levels of Borrelia burgdorferi indicates this has a prevalence peak in April and May, at 55oC and Bartonella henselae peaks around June and July when it is warmer. Variability in the permanence of those pathogens within the ticks.

DNA Sequence revealed:

In a manuscript for future publication in Journal of Clinical Microbiology we firm up the data to sequence what we’ve got, sequence to find which Mycoplasma subtype it is.
In the first tick there is a 93% homology to Mycoplasma fermentans and we were very excited. Clinical data and the tick information we had tied in.
In second tick, date for Mycoplasma fermentans also correlation with the tick carrying this bacterium. In second, 95% correlation.

So there is a correlation by independent study in a patient between what the tick is carrying and what is in the patient.

Conclusions:

In addition to Borrelia burgdorferi, Babesia microti and Erlichia, molecular evidence exists for the presence of Bartonella henselae and Mycoplasma in the Ixodes ticks in New Jersey.
This is the first report of a black leg tick harbouring Mycoplasma.
Infection of ticks with two or more bacterial strains occurred in approximately 25% of those assayed.
Seasonal effects were evidence in which Borrelia burgdorferi prevalence rates peak in April and May, wheras Bartonella henselae peaks in June and July.
We don’t know about ticks in Europe - our lab would love to have the information.

I won’t go through this part of my talk about a new technique. Briefly, its about a new real time PCR, the new generation of PCR testing. Real time PCR and pyrosequencing for Lyme Disease (title).

In conventional PCR, we amplify a specific amplicon of the genome within the target of interest. (Bb, amplilfy a specific gene in the bacterium – can tehn identify it).
RTPCR is more sensitive and more specific, while we amplify we see it in real time, so it is much more sensitive. RTPCR benefits are:
Increased sensitivity and
Increased specificity and
gives a shorter turn around time
we can quantitate the result.

Another aspect of our lab work is the pyro-sequencing procedure. In this we are a pioneering laboratory in the USA. It’s a techniquie in which you can amplify a specific region within a bacterium or a virus (e.g. if this was an antibiotic resistance region) eg Mycoplasma and give the Levofloxacin to a patient. - This patient’s infection resists Levofloxacin, how can we find out this resistance, but we know Levofloxacin is active, so take this specific region (that Levofloxacin binds to) sequence it at where the gyrase binds and can find out within 30 minutes the exact location where we have a mutation in this Mycoplasma. We can find isolates that are resistant to antibiotics.
You can find this within half an hour. Can use the same technique to find different genotypes within the ticks. We can tell them all apart.

This is again, same technique, OspC Pyrosequencing identification of Borrelial species. (Re: the Borrelial species (burgdorferi, afzelii and garinii).

To distinguish between Bb and the European strains. European strains Afzelii and garinii. Why is it important to differentiate? Both these strains are more virulent and more associated with neuroborreliosis than Borrelia burgdorferi, wheras B. burgdorferi (US strain) is more associated with arthritis. It’s important to know the variations.

Way we can see that is if I align spot in - red, blue, green is where they change, you can find regions where they look alike and we can amplify them and amplify the regions where they don’t look alike. And that is how we tell the difference between one and another. . Can differentiate between the species.

So, Uses of Pyrosequencing are: genus and species identification, secondly to find point mutations/deletions/insertions and lastly to find antibiotic resistance. Thank you.