Improved Borrelia burgdorferi Blood culture Method

Medical topics with questions, information and discussion related to Lyme disease and other tick-borne diseases.
nnecker
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by nnecker » Tue 19 Feb 2013 23:48

Ticksuck let me ask you this:

[quote]Lorima wrote from Persistent Spirochete Antigen In Mice:

I guess I've gotten into the habit of ignoring Lyme publications from Yale. Not that it's uninteresting from a political point of view to see what they're saying, just that time is limited, and once I've seen a number of papers indicating a unanimous institutional bias from a place, it feels like a waste of time to read the stuff that comes out of there.[quote]

Shouldn't Lorima let the subject of a legitimate research paper being reported, with important results which should stand on it's own, instead of focusing on biases and the association with Yale University?

I didn't hear anything about "shill'in" from you then.

TicksSuck
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by TicksSuck » Wed 20 Feb 2013 1:54

nnecker wrote:Ticksuck let me ask you this:
Lorima wrote from Persistent Spirochete Antigen In Mice:

I guess I've gotten into the habit of ignoring Lyme publications from Yale. Not that it's uninteresting from a political point of view to see what they're saying, just that time is limited, and once I've seen a number of papers indicating a unanimous institutional bias from a place, it feels like a waste of time to read the stuff that comes out of there.

Shouldn't Lorima let the subject of a legitimate research paper being reported, with important results which should stand on it's own, instead of focusing on biases and the association with Yale University?

I didn't hear anything about "shill'in" from you then.
Oh my, nnecker! You really don't have anything intelligent to say, do you?

TicksSuck
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by TicksSuck » Wed 20 Feb 2013 22:38

inmacdonald wrote: Alan MacDonald MD : Comment:
...
All patients are those with Chronic Persistent Lyme Borreliosis who have undergone long term antibiotic therapy and who were off any antibiotics for a minimum of 6 week prior to the procurement of the blood cultures
...
The implications for Treatment are clear.
The implications for Diagnosis are Clear.
This characteristic of the patients has not been described in the referenced paper. Would you have any insight as to the reason why, if indeed it was a characteristic?

Camp Other
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by Camp Other » Sat 23 Feb 2013 2:57

TicksSuck wrote:
inmacdonald wrote: Alan MacDonald MD : Comment:
...
All patients are those with Chronic Persistent Lyme Borreliosis who have undergone long term antibiotic therapy and who were off any antibiotics for a minimum of 6 week prior to the procurement of the blood cultures
...
The implications for Treatment are clear.
The implications for Diagnosis are Clear.
This characteristic of the patients has not been described in the referenced paper. Would you have any insight as to the reason why, if indeed it was a characteristic?
I agree, this would be important information to share. If this is so, why was it left out?

Camp Other
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by Camp Other » Sat 23 Feb 2013 3:00

nnecker wrote:Ticksuck let me ask you this:
Lorima wrote from Persistent Spirochete Antigen In Mice:

I guess I've gotten into the habit of ignoring Lyme publications from Yale. Not that it's uninteresting from a political point of view to see what they're saying, just that time is limited, and once I've seen a number of papers indicating a unanimous institutional bias from a place, it feels like a waste of time to read the stuff that comes out of there.

Shouldn't Lorima let the subject of a legitimate research paper being reported, with important results which should stand on it's own, instead of focusing on biases and the association with Yale University?

I didn't hear anything about "shill'in" from you then.
I think it's important to look at all the research, including Yale's. And in doing so, it's important to also ask questions about what any papers omit that would be important to know in assessing the outcomes as well as look at methodology and de facto assumptions made by the authors of such papers.

Lorima
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by Lorima » Sat 23 Feb 2013 5:48

Regarding Sapi et al:

This is a very good paper. I'm happy to see someone doing good research on LD testing, and making progress. I will carefully check any serious critiques that are made of it, to see if there is merit in them. Even if they come from Yale. ;)

I expect there will be more papers from Sapi's group, and there will be more details about various subsets of patients in them. I don't know how soon this will be; but I won't complain if we have to wait. In science, quality is more important than quantity of publications.

This particular article is devoted to describing the method. I was waiting to see if it seemed plausible that this new method was such an improvement, and I think it is plausible. The addition of the collagen step was smart, and logical, because it is well known that Bb has an affinity for collagen. It paid off. 

The paper has plenty of data; I don't think it would have been helpful to add patient characteristics. It would certainly have been unwise to add patients who were not CDC-positive but were probably infected. That would have muddied the waters by introducing too many variables. When one is developing a laboratory method, it's much better to keep things well-controlled. Simply using CDC-positive vs. CDC-negative patient samples, satisfies the requirement for comparing any new method to the "gold standard" 2-tier test, without introducing extraneous side-issues. 

The title and the conclusions are modestly stated, and the abstract matches what is in the paper.

Note the two sets of patients; one to optimize the method (the first 50), and then another set of 72, to see if the method derived using the first 50, holds up. 

The spiked samples provide critical quantitative information, and the reporting of variations that were tried, and which ones worked the best, are important for appreciating what has been optimized, versus what might respond to future tweaking. I think a lab staffed by technically experienced and talented hands could repeat this, but it wouldn't be likely to succeed in less meticulous hands. There are a lot of steps, and no room for sloppiness or haste.

Finally, the paper is lucid. You can read through it with a minimum of pulling up other papers to figure out exactly what was done, or where assertions came from. 

It would be nice to know where your personal strain fits into the phylogenetic tree, wouldn't it? My family patients are on antibiotics now, or I'd send off some samples. It doesn't seem overpriced to me, considering the labor and the skill involved.
Int J Med Sci 2013; 10(4):362-376. doi:10.7150/ijms.5698 

Improved Culture Conditions for the Growth and Detection of Borrelia from Human Serum

Eva Sapi1,2 , Namrata Pabbati1, Akshita Datar1, Ellen M Davies1, Amy Rattelle1, Bruce A Kuo1

1. Research Division of Advanced Laboratory Services Philadelphia PA, USA;
2. Department of Biology and Environmental Science, University of New Haven, West Haven CT, USA.

Abstract: 

In this report we present a method to cultivate Borrelia spirochetes from human serum samples with high efficiency. This method incorporates improved sample collection, optimization of culture media and use of matrix protein. The method was first optimized utilizing Borrelia laboratory strains, and later by demonstrating growth of Borrelia from sera from fifty seropositive Lyme disease patients followed by another cohort of 72 Lyme disease patients, all of whom satisfied the strict CDC surveillance case definition for Lyme disease. The procedure resulted in positive cultures in 47% at 6 days and 94% at week 16. Negative controls included 48 cases. The positive identification of Borrelia was performed by immunostaining, PCR, and direct DNA sequencing.

Materials and Methods: 

snip

Blood collection

Full informed consent was obtained from each study participant and HIPAA-compliant privacy measures were maintained. All patients who participated in this study had not been exposed to antibiotics for a minimum of 4 weeks prior to blood sample collection [33] [34]. Sera from 50 seropositive patients were used to establish the culture media and conditions. After the cultivation parameters were established, sera from an additional 72 Lyme disease patients (26 males and 46 females ranging in age from 3 to 80 years with average of 42 years) were collected between March 2011 and May 2012 and used to evaluate this culture method. All of these patients had a clinical presentation consistent with Lyme disease and their illness was confirmed by laboratory evidence of infection using the Center for Disease Control (CDC) recommended, 2-tiered serological method, thus they all satisfied the strict CDC surveillance case definition for Lyme disease.

To determine the specificity of the test we collected serum from 48 healthy controls that had no history of either tick-bites or Lyme disease (21 males and 27 females ranging in age from 27 to 71 years with an average of 43 years). Patients and controls were selected from private medical practices from 15 different States (CA, AR, CT, NH, NJ, NY, MA, MD, MN, OR, PA, TN, UT, VT). Samples were delivered to the laboratory and cultures were initiated within 24-30 hours of collection. 

Culture protocol

Blood samples (~30 ml /patient) were collected in the following tubes: 9.0 ml peripheral blood in a 15.0 ml polypropylene Falcon tube containing 5 ml BSK-H, two samples consisting of 9.0 ml peripheral blood in 10.0 ml red top (no additive) Vacutainer tube, or Vacutainer tubes containing EDTA (purple top). Blood samples were transported to the laboratory overnight at room temperature and upon receipt they were allowed to sit undisturbed for several hours at room temperature to allow serum separation. The serum was divided into eight starter cultures for each clinical sample and mixed with modified BSK-H media (mBSK-H) - four in the standard 15 ml glass tube (13 ml final volume) and four in the small 2ml cryo tubes (1.8 ml final volume) as summarized in Table 2. Cultures were incubated at 34º C with 5% CO2 and 100% humidity. The lids of all culture tubes were closed loosely to allow limited gas exchange between the culture medium and the environment. Starter cultures were harvested after 6 days and used for either immunocytochemical studies or to seed a long-term culture. Long-term cultures were established by combining and directly transferring cells and media from culture tubes to a Coplin jar with an additional 15 ml mBSK-H media (total volume of 34 ml), a 5 µg rifampicin disc (BD Scientific) and 2 collagen coated slides (Rat-tail type I; BD Scientific) and maintained through 16 weeks at 34oC with 5% CO2 and 100% humidity. After 8 weeks, one slide was checked under dark field microscopy and used for polyclonal anti-Borrelia antibody detection analysis. If the fluorescent antibody result was negative at 8 weeks of culture, another collagen coated slide was placed in the Coplin jar and half of the culture media (~17 ml) was replaced with fresh mBSK-H medium and the culture was incubated for an additional 8 weeks. 

snip

Results

snip

In infected mammals, Borrelia are most often found to be sequestered in collagen and especially collagen type I [47] [48]. A collagen matrix has been shown to support growth of Borrelia burgdorferi B31 and 297 strains in vitro [38] [49]. To study this further we initiated an experiment in which different amounts of Bb B31 cells (1, 10, 100, 1000) were spiked into 10 ml of whole peripheral blood from healthy donors and after a short term culture of 6 days in mBSK-H the culture was transferred to long-term cultures set up in Coplin jars with collagen-coated slides in a total of 45 ml of mBSK-H media. After 14 days, an accumulation of Borrelia growth was observed on all collagen coated slides. To evaluate this growth, a slide was removed from the Coplin jar and stained with the BacLight Live/Dead staining kit (see Materials and Methods). Figure 3A demonstrates a large thriving colony of attached spirochetes, with a very low proportion of non-viable spirochetes. Blood samples that were not spiked with Borrelia cells did not produce any spirochetes on the collagen-coated slides (Figure 3B). These data confirm that under favorable conditions human blood containing as few as one Borrelia B31 spirochete can be cultured in mBSK-H with a collagen source, which supports the original hypothesis that a matrix-supported environment is beneficial for their growth.

Validation study in 72 CDC-positive Lyme disease patients

This study enrolled 72 Lyme disease patients whose diagnosis was confirmed by a typical clinical history and a positive result on a 2-tiered serological test following CDC testing guidelines. It was required that each donor had not been exposed to antibiotics for a minimum of 4 weeks prior to blood draw as recommended in previous studies [33] [34]. Short-term cultures were established in mBSK-H as described and after six days these cultures were used to seed collagen-supported long-term cultures. The details of the procedure are summarized in Figure 5. Analysis on day 6 using polyclonal anti-Borrelia antibody revealed that 34 of the 72 (47%) cultures showed growth (Figure 6). Long-term cultures were then begun from all the patient samples at day 6 and were analyzed again at 8 and 16 weeks (Figure 6). An additional 26 samples became positive for Borrelia at 8 weeks for a total of 60 of 72 (83%) and at 16 weeks an additional 8 samples became positive for Borrelia 68 of 72 (94%). All samples that were positive by polyclonal staining (representative images in Figure 7) also stained positively with the monoclonal antibody (representative images in Figure 8). 

Borrelia-specific PCR products were amplified from each of the antibody-positive samples confirming that each sample contained Borrelia. Sequence analysis at the 16S rRNA or CTP synthase locus or both was performed and confirmed that each PCR product was Borrelia DNA. Significant sequence variations were identified among 51 samples sequenced at the CTP synthase gene locus indicating that samples were derived from independent sources and not from laboratory contamination. The percentage of the sequence identity of the clinical isolates to the Borrelia burgdorferi B31/N40 strains reference sequences (accession #AE000783 and #CP002228 respectively) ranged from 92%-100%. The numbers of polymorphic sites found in the clinical isolates for CTP synthase loci and the percentage of identity to Borrelia burgdorferi B31 sequence summarized in Table 3. 

snip

Discussion

snip

Several measures were employed to rule out the possibility of false positives. Blood from 48 negative controls was cultivated using the above established methods and none showed any growth. Meticulous DNA analyses of the positive cultures including the use of both negative and positive controls in each PCR reaction did not reveal any evidence for contamination. To confirm that Borrelia isolates that were cultured from the 72 CDC positive patients were unique and to further prove that the PCR products were not from an environmental contaminant, PCR products were directly sequenced at either the 16S rRNA or the CTP synthase locus or both. The results confirmed that the sequences from each positive culture sample were derived from Borrelia. Sequence analysis within the amplified region of 16S rRNA gene showed only limited variation that is likely due to the highly conserved nature of 16S rRNA gene [55]. However, abundant sequence variations were noted at the CTP synthase locus from the positive cultures, indicating that the clinical isolates are unique and derived from wild-type Borrelia and not from a laboratory contaminant. The PCR and sequencing results were included in this study to further validate the polyclonal and monoclonal antibody assays used for the development of this Borrelia culture.

In summary, this report provides evidence for the value of a novel method for culturing Borrelia from human serum samples. The inclusion of key components such as modified culture media plus a unique culture environment has resulted in an improvement in the ability to cultivate this organism. This new culture method directly addresses the issue of the low numbers of Borrelia in clinical samples by amplifying their quantity through long term culture in which Borrelia were able to thrive for as long as eight months (data not shown). The versatility of this method allows for samples to be harvested from the culture at any point in time for further study, and it also serves as a source of Borrelia for a variety of direct detection techniques as well as for additional research. Finally, this unique culture method could play an important role in providing useful diagnostic information for select Lyme disease patients who might have tested negatively by other methods. 
"I have to understand the world, you see."
Richard Feynman

RitaA
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by RitaA » Sat 23 Feb 2013 7:47

Lorima,

Thanks very much for that wonderful analysis. I do hope the improved blood culture method lives up to expectations, and so far it appears there's no scientific reason that it won't.

This may not be a perfect transcript of what Marty Schriefer * said during the September 24th, 2012 Webinair "HHS Federal Research Update on Lyme Disease Diagnostic Activities", however it's close enough for my purposes (for more about the FDA Webinair, see http://www.lymeneteurope.org/forum/view ... 224#p34549 ):
The benefit of culture is you have the agent in the laboratory. It's really a gold standard. But the downside is that spirochetes are slow growing, take days to weeks to detect.

... I think we may see culture coming back, particularly in early stage, perhaps in Stage 2 disease as a diagnostic tool. But currently it's of limited diagnostic application.
More from Marty Schriefer as to the limitations of current blood culture methods:
... If we move then into cases that have neurologic involvement or arthritic involvement, culture is rarely successful in these cases and only anecdotally has been reported in that neurologic and joint involvement.

In contrast, PCR has had some realized sensitivity in both of these manifestations of Lyme disease. And again I'm looking forward to seeing a greater utilization of PCR as a diagnostic tool in the future.
I must admit that I'm a bit confused because I've read conflicting views when it comes to PCR, including this -- chosen for its plain English:

http://www.medscape.com/viewarticle/742847
Medscape Medical News

PCR Not a Reliable Test for Active Lyme Arthritis

Janis C. Kelly
May 17, 2011

May 17, 2011 — Polymerase chain reaction (PCR) testing for Borrelia burgdorferi DNA can confirm the diagnosis of Lyme arthritis but is not a reliable indicator of active infection in the synovial fluid of patients whose arthritis persists after antibiotic therapy, according to a new study.

Researchers suspect that persistence of positive PCR results in such cases is the result of an autoimmune response to moribund or dead bacteria and recommend treatment with disease-modifying antirheumatic drugs (DMARDs).

[snip]

"Our results confirm that detection of the bacteria which causes Lyme disease in synovial fluid is not a reliable test of active joint infection," Dr. Steere concluded. "We recommend treatment of patients with Lyme arthritis with appropriate oral and if necessary, IV antibiotics for two to three months. However, in those with persistent arthritis despite oral and IV antibiotics of this duration, we then give DMARD treatment."

"The study has been carefully done and represents an important contribution to our understanding of Lyme arthritis," Gary Wormser, MD, chief of infectious diseases at New York Medical College in Valhalla, told Medscape Medical News. He was not involved in the study.

"From the clinician's perspective, the utility [of PCR] seems quite limited at this point in patients who have received recommended treatment and recommended retreatment according to Infectious Diseases Society of America...guidelines," Dr. Wormser added. "It would be desirable, however, to expand the database on this issue, as the above study was relatively small. I think that doing PCR posttreatment is more of an option than a requirement. Some might feel more secure in starting certain DMARDs in PCR-negative cases."
I think it's fair to say that Marty Schriefer* and Drs. Steere and Wormser are not in agreement when it comes to the usefulness of PCR testing.

It will be interesting to see what objections (if any) certain individuals might have to the improved blood culture method. I agree that any attempt to replicate the results of Sapi et al should be carried out by a research team whose qualifications and objectivity are beyond reproach. In the current atmosphere of controversy and conflict, it would have to be a research team that everyone could trust, and that's a mighty tall order these days.

* Marty Schriefer = Dr. Martin Schriefer - Chief, Diagnostic and Reference Laboratory, Division of Vector-Borne Diseases, CDC Reference Reagents for Development and Evaluation of Lyme Disease Diagnostic Assays

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inmacdonald
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by inmacdonald » Sat 23 Feb 2013 14:20

For Comparison - A European Report - 70 consecutive positive Blood Cultures of Borrelia burgdorferi Sensu lato

 Ruzic, E  Arnez,M     J Medical Micro 50(10) 896-901

Link:
http://jmm.sgmjournals.org/content/50/10/896.long
European Patient Study Group:::
All cases from early LD - all EM positive


A positive blood culture recovery rate in seventy consecutive 
LB cases has, to my knowledge, never been  equaled  in
any USA center.
I  did not publish my blood culture method from the 1980' s
with Joe Burrascano's patients and my modification of Base BSK
The total number of Joe's patients which we
studied together was 76 positive borrelia blood cutures as I recall.
In the study with Dr Bernared W.Berger of Skin punch biopsies obtained with
sterile surgical technique from Erythem Migrans advancing edge junction of
erythema migrans with non erythematous Skin---
a total of 63 cases were processed :: Bb = cultures from  skin  in year 1986 =9 POSITIVE:::
:::Skin Biopsies in year 1987 =7 POSITIVE :::: Contaminated Biopsy Cultures ::1986= 2 cases
,:: 1987=7 cases, no growth at all from EM Skin Biopsy cultures N=47.
Ratification of the Borrelia burgdorferi Isolates from Erythema MIgrans punch biopsies
was performed at NIH NIAID,Rocky Mountain Lab, Hamilton Montana by
Osp A (31 kd) immunostaining of SDS products of solubilized Borrelia burgdorferi skin
cultures by MacDonald::
ALL 8 ISOLATES were POSITIVE for Monoclonal Antibody+ H5332
Intensity of staining in OspA [H5332 band]
 isolates 1,4,5,6,7,8 =1+ positive bands all equal in intensity
isolate 2 - 2+ positive staining,
isolate 3= 3+ staining.
Today, all isolates would be DNA sequenced for a  reasonable
diagnostic mer  length and deposited at the American Type Culture Collection {ATCC]

See my paper " Clinical implications of delayed growth of Borrelia burgdorferi"

https://www.dropbox.com/s/bpola7so4kpki ... ec1990.pdf

which appeared in Acta Tropica coauthored with Dr Tom Schwan and Dr Bernard Berger.
Tom Schwan verified 8 isolates out of a total of 16  submitted to Rocky Mtn. Lab  as Bb, but with one
of the eight showing a +++ (unusually prominent 
24 kd band  ( OspC? co-expressed with Osp A?),::
isolates 6 and 7  showing a + 24 kd protein band) on SDS protein profile.
Dr. Schwan elected to study only 8 of the 16 positive cultures with OspA
gel electrophoresis, and these were selected randomly from the 16 total submitted
as a reasonable sample of the submissions for verification of the Exact species
of Borrelia as Borrelia burgdorferi. 
I also submitted  all isolates from my work to Professor Russell Johnson At Univ, of Minnesota. 
All were confirmed as Borrelia burgdorferi.
None of the Skin culture isolates were "cookie cutter clones of B31".
link:  to published paper demonstrating the protein bands of the Skin culture isolates from Erythema Migrans skin
biopsy cultures: Acta Tropica website: Full text reproduced below


Conclusion:
In spite of Dr Jorge Benach's statement in the thread above this post
describing a 5% expected blood culture yield of Borrelia burgdorferi isolation
there are now TWO independent reports of SEVENTY consecutive Borrelia burgdorferi
isolates from human Blood
.

This pair of reports :: One from Europe and One from The USA written more than a decade apart
reinforces the validity of Blood culture [Primary isolation } of Borrelia burgdorferi from human blood
in patients with Lyme Borreliosis manifesting as either erythema migrans or by LB symptoms and previous
positive LB serologies.
_____________________________________________________
The Europeans found both B. afz and B. Gar in their series, 
so heterogeneity  in a series of + cultures from the "wild" 
adds rather than subtracts  support as "reality" and within expected
previously posted human studies of Bb culture of blood or solid tissue.

We all agree that Bb is never "normal flora " in any human at any time.
We can also agree that the Advanced Labs report has in effect been replicated
by a previous group using a different culture medium from the European Community.
Respectfully,
Alan B.MacDonald MD

nnecker
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by nnecker » Sat 23 Feb 2013 14:45

Lorima wrote on Jul 4,2012:

I've gotten into the habit of ignoring Lyme publications from Yale.......... it feels like a waste of time to read the stuff that comes out of there.

Lorima wrote on Feb 23,2013:

I will carefully check any serious critiques that are made of it, to see if there is merit in them. Even if they come from Yale.


It is so nice of you to conveniently change your mind about Yale Lorima. :oops:

duncan
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Re: Improved Borrelia burgdorferi Blood culture Method

Post by duncan » Sat 23 Feb 2013 15:40

nnecker, let me take a stab here: English not your native language? You have to bring a certain tolerance of nuance and intonation to appreciate it sometimes. So if it is a second language for you, in the future you may wish to enlist help in translation to avoid any unseemly confusion. ;)

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