Friday, May 27, 2022

Dark Field Microscopy Lyme Disease

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Major Red Flag: Unscientific Medical Practitioners

Lyme Spirochete After Antibiotic – Dark Field Microscopy Live Blood
  • Including those who market themselves using the following terminology: Lyme literate , integrative, functional, alternative, complementary, Traditional Chinese Medicine, holistic, natural, Biological, Ayurvedic, chiropractic, naprapathic, homeopathic, anti-aging, regenerative , environmental , and naturopathic.
  • Including members of organizations listed as questionable by Quackwatch, such as the American Academy of Environmental Medicine.
  • A surprising number have had a personal experience where themselves or a loved one received a fictional diagnosis.
  • They frequently profit from sales of unproven or disproven treatments like herbs and supplements, a practice that naturopathic whistleblower Britt Hermes deemed unethical.

There arent many absolutes in medicine but one is never trust a doctor who has their own line of dietary supplements.

James Hamblin

Detection Of Pleomorphic Borrelia Forms In Vivo

Brains of three patients with pathologically and serologically confirmed Lyme neuroborreliosis and concurrent AD were analyzed . From the brains of these three patients, aged 74, 78, and 86 years, spirochetes were successfully cultivated in BSK II medium. In two of them 16SrRNA gene sequence analysis identified the spirochetes as Borrelia burgdorferi sensu stricto .

To detect whether atypical and cystic forms of Borrelia burgdorferi spirochetes are present in the brains of these patients, frozen sections of the hippocampus, frontal, temporal and parietal cortex were analyzed using dark field microscopy, as well as histochemical and immunohistochemical techniques. Before immunostaining the sections were fixed in acetone for 10 minutes at 4°C. Acetone fixed frozen sections which were cut from samples taken from identical brain areas in three control patients without neurological symptoms and without brain lesions were also processed and analyzed in the same fashion.

Paraffin sections cut from various cortical samples of two patients with clinically, serologically and pathologically confirmed general paresis, were also analyzed for the presence of Treponema pallidum. The goal was to compare atypical spirochetal forms of Borrelia burgdorferi in Lyme neuroborreliosis with those of Treponema pallidum in general paresis.

Techniques For Epidemiological Study

Molecular typing is used for species identification of Borrelia in epidemiological studies . In Ethiopia, molecular investigation of 284 cattle ticks found a potential new Borrelia species distinct from both the relapsing fever group and Lyme borreliae . In Ethiopia, B. recurrentis DNA has also been found in 23% of head lice from patients with louse-borne relapsing fever .

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Influence Of Ospa/b Deficiency On Bb Infectivity For Mice

To examine a potential role for OspA/B in the infectious phenotype of Bb, groups of five C3H/HeN mice were inoculated intradermally with varying numbers of either the OspA/B-deficient mutant or wild-type 297. 2 wk after injection, ear punch biopsies were harvested and cultured in BSK-H medium . Both wild-type 297 and OspA/B spirochetes were readily observed in all cultures of ear punch biopsies, indicating that wild-type 297 and the mutant had similar infectious phenotypes. Further analyses indicated that the OspA/B mutant maintained its mutant genotype and phenotype after recovery from mice . To further assess tissue dissemination by the OspA/B mutant, we next used quantitative PCR for flaB as a surrogate indicator of spirochete loads in mouse skin, joints, and hearts. Mice infected with the OspA/B mutant had similar or even higher levels of spirochetes when compared with mice infected by wild-type 297 . Further analysis of joint swelling and histopathology revealed that the OspA/B mutant elicited arthritis more pronounced than that induced by wild-type 297 , perhaps as a consequence of the higher numbers of spirochetes detected in joints . Nonetheless, the combined data indicated that OspA/B function is not essential for Bb infection, replication, dissemination, or pathogenesis in the murine model of Lyme borreliosis.

Cultivation Of Borrelia Burgdorferi Spirochetes In Bsk Ii Medium

Live Blood Microscopy Dark Field

Borrelia burgdorferi spirochetes strains B31 and ADB1 were cultivated in BSK II medium . To 500 ml BSK medium containing 6% rabbit serum and 7% gelatin , 6 mg acetyl muramic acid and 0.2 g N-acetyl glucosamine , Rimactan and Fosfocin were added. The spirochetes were cultivated at 32°C. The pH of BSK II medium was adjusted to pH 7.

To induce atypical spirochete forms 5 ml of cultivated Borrelia burgdorferi spirochetes were exposed to various harmful conditions. Spirochetes were exposed to strong acidic and basic conditions by adjusting the pH of the BSK II medium to pH2 and pH10 using sterile 1 M HCl or 1 M NaOH. The harmful effect of alcohol was analyzed by adding 1 ml of either 70% or 95% ethanol to 5 ml cultivated spirochetes. Heat shock was produced by cultivating spirochetes at 45°C.

Spirochetes are known to bind Congo red and Thioflavin S , both of which are widely used to stain amyloid. To analyze whether they may induce atypical Borrelia forms, 1 mg or 5 mg of Congo red, or 1 mg or 5 mg Thioflavin S were directly added to 5 ml of spirochete culture. The same amounts dissolved in 2 ml of 70% alcohol were also used to induce atypical spirochetes. The effect of acridin orange, another fluorochrom which binds to spirochetes, was also analyzed by adding 1 mg or 5 mg acridin orange powder to 5 ml of cultivated spirochetes.

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Detection Of B Burgdorferi Antigens

Given the difficulties of cultivating or detecting spirochetes by staining, considerable interest has been directed at developing alternatives for the detection of spirochetes. One approach has been to identify specific antigens of B burgdorferi in the urine of patients suspected of having Lyme disease. Several groups have already shown that Lyme spirochetes may be present in the urine of naturally infected white-footed mice, Peromyscus leucopus , and that the urinary bladders of both experimentally and naturally infected rodents often produce spirochetes when the organs are triturated and inoculated into BSK II medium . Antigens of B burgdorferi, including outer surface proteins A and B and the flagellar protein, have been detected in the urine of both laboratory mice infected with B burgdorferi and humans with Lyme disease . Urine containing these antigens was filtered through a membrane that was then incubated with one of three specific monoclonal antibodies in a dot blot immune assay.

Although the initial results were encouraging and one urine-based antigen test was marketed commercially for a brief time, no such test for confirming Lyme disease in the laboratory is currently available.

Detection Of B Burgdorferi Using Dna Probes And The Polymerase Chain Reaction

In the past few years, another strategy developed at Rocky Mountain Laboratories to detect B burgdorferi has been to demonstrate the presence of DNA specific to this spirochete. First, hybridization probes were developed to identify specifically both purified DNA of B burgdorferi and whole spirochetes . Although the probes specifically identified Lyme spirochetes, the sensitivity of some probes was unsatisfactory because detection required a minimum of 10,000 spirochetes. Subsequently, assays have been developed using the polymerase chain reaction. These assays are specific for B burgdorferi, capable of detecting as few as one to five spirochetes, and able to type spirochetes from North America and Eurasia into two groups .

Other workers have subsequently developed further polymerase chain reaction assays amplifying other regions of the spirochetes genome to identify B burgdorferi DNA and to detect the spirochete in fresh ticks , ticks preserved in alcohol , or the urine of human patients with Lyme disease .

Polymerase chain reaction for the laboratory confirmation of Lyme disease is not yet commercially available.

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Dark Field Microscopy Histochemical And Immunohistochemical Analysis Of Spirochetes

From cultivated Borrelia spirochetes exposed to various harmful conditions, 50 l samples were used as wet preparation for dark field microscopy analysis. Additional samples were used to prepare smears for the histochemical and immunohistochemical analyses. In order to analyze free floating spirochetes in Borrelia infected cell cultures, 50 l samples of the co-culture medium were analyzed.

Floating paraffin sections of the cerebral cortex of the two patients with general paresis were immunostained with a polyclonal anti-Treponema pallidum antibody .

Infection Of Cell Cultures With Borrelia Burgdorferi

Dark Field Microscopy – Blood 800X Spirochete Lyme Blebbing or Cyst Transformation To Pearls

Superior cervical ganglia from 8- to 12-day-old chicken embryos were dissociated as described previously . Briefly, neurons were separated from non-neuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 34 weeks in serum containing medium on a polyornithine substrate pre-coated with heart-conditioned medium . Neurons dissociated from the telencephalon of 21-day-old rat were cultured either on collagen or polylysine substrate in a serum-containing medium . Rat primary astrocytes were prepared as described earlier . Following the characterization of the primary astrocytic cell cultures using anti-GFAP antibody more than 95% of the cells were GFAP reactive . The cells were cultured in 2 well chambers or in six well clusters in a humidified CO2 incubator at 37°C.

To infect neurons and astrocytes, Borrelia spirochetes of the virulent strains B31 and ADB1, the latter having been cultured from the brain of a patient with concurrent Lyme neuroborreliosis and AD , were employed. Equal volumes of medium for the given primary cells and for spirochetes were used as the culture medium. The final concentration of spirochetes in the infected medium corresponded to 5 × 105/ml. Before exposure to spirochetes, the cells were tested with 4′,6-diamidine-2′-phenylindole dihydrochloride to exclude Mycoplasma infection. Parallel cultures not infected with spirochetes were always used as controls.

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Characteristics Of Borrelia Burgdorferi

Borrelia burgdorferi is a helical-shaped spirochete that is poorly gram-stained . It is better viewed under a dark ground microscope or by silver impregnation staining. The ultrastructure of Borrelia is similar to Treponema and Leptospira with minor differences.

  • Size: Larger, 10-30 m in length and 0.2-0.5 m in width.
  • Spirals: They are less in number with wider spirals and longer amplitude
  • Endoflagella: More in number , attached subterminally at the pole.

Lyme Borreliosis: Disease Spectrum

Infection with B. burgdorferi sensu lato can result in dermatological, neurological, cardiac, and musculoskeletal disorders. The basic clinical spectra of the disease are similar worldwide, although differences in clinical manifestations between LB occurring in Europe and North America are well documented . Such differences are attributed to differences in B. burgdorferi sensu lato species causing LB on the two continents. Furthermore, differences in clinical presentations exist between regions of Europe, presumably due to differences in the rates of occurrence of infection caused by distinct B. burgdorferi sensu lato species .

Patients with B. burgdorferi sensu lato infection may experience one or more clinical syndromes of early or late LB. Usually, early infection consists of localized erythema migrans , which may be followed within days or weeks by clinical evidence of disseminated infection that may affect the skin, nervous system, heart, or joints and subsequently, within months, by late infection . Arthritis appears to be more frequent in North American patients , whereas lymphocytoma, acrodermatitis chronica atrophicans , and encephalomyelitis have been seen primarily in Europe .

While Lyme arthritis is the most common late manifestation of LB in North America, ACA appears to be the most common manifestation of late LB in Europe. As mentioned earlier, these differences are likely due to the different species causing LB in the two continents .

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Major Red Flag: Chronic Lyme Where Real Lyme Is Rare Or Non

Chronic Lyme is a social phenomenon spread by word of mouth and the Internet. Thus, chronic Lyme is not restricted to areas where ticks transmit the curable Lyme infection.

With real Lyme disease, 95% of reported US cases are in 14 states, mostly in the Northeast, mid-Atlantic, and Upper Midwest.

Healios excellent article on the misnomer of chronic Lyme disease features this graphic illustrating the stark difference in geography between confirmed Lyme cases and chronic Lyme support groups:

The United States has exported its conspiracy theories about Lyme disease to many countries, including Canada, France, Germany, and Turkey. Thousands of people have received false diagnoses in Australia, where there is no endemic Lyme disease.

What Is Wrong With Microscopy In Borreliosis

Dark Field Microscopy
  • What is wrong with microscopy
  • Nothing, as you will see from the below evidence.

    Microscopy in diagnostics

    Microscopy played a great role in identifying unknown pathogens and it is still the first line of diagnostics in some diseases where the novel diagnostic tools failed first time or did not prove to be efficient in the long term. Examples are syphilis and tuberculosis, less specifically, the scanning for urinary tract infections is done by automated microscopy or unknown genital infections are identified manually.

    Even when methods such as isolation or cultivation of bacteria is mentioned, you must know that this includes some type of microscope at the end, when the pathogens are identified.

    Pathogens are most of the time invisible even under a microscope, and therefore the contrast needs to be increased against the background. The easiest method is staining with various agents such as non-specific or specific stains .

    Historic discovery and diagnosis of spirochetes

    Since 1909, examinations of unstained and unfixed preparations by dark-field microscopy have been able to show with complete certainty the presence of infection caused by Spirochetes, even before the appearance of antibodies . This is the point when dark-field microscopy entered the area of routine diagnostics.

    It is important to mention that Borrelia burgdorferi was first identified by dark-field microscopy, and it is still routinely used in scientific experiments when Borrelia are isolated .

    References

    Recommended Reading: My Dog Tested Positive For Lyme Disease

    Complementation Of The Ospa/b Mutant

    To complement the OspA/B mutant with a wild-type copy of ospAB, a 2-kb DNA fragment containing the wild-type ospAB genes first was PCR amplified from Bb strain 297 using primers 3 and 4 . This fragment subsequently was cloned into pCR-XL-TOPO , resulting in plasmid pXT-ospAB-2kb. pXT-ospAB-2kb was then digested with SacI and XbaI, and the 2-kb fragment containing the ospAB genes was inserted into the corresponding sites of pBSV2 . The resulting plasmid was designated pOspA/B . To complement the OspA/B mutant with only wild-type ospA, pOspA/B was digested with AspE1 and BstAp1 . The linear plasmid with the ospB deletion was then blunt ended and ligated closed, culminating in plasmid pOspA. For complementation, 20 g of pOspA/B or pOspA DNA was electroporated into the OspA/B mutant, and 20 kanamycin-resistant/Strepr clones were obtained. To confirm that these clones contained the pOspA/B or pOspA plasmid, whole cell lysates of kanamycin-resistant/Strepr cells were used to transform TOP10 E. coli. Plasmid was then rescued from these E. coli transformants, and restriction digestions were performed to verify recovery of the respective plasmid.

    Essential Role For Ospa/b In The Life Cycle Of The Lyme Disease Spirochete

    The online version of this article includes supplemental material.

    X.F. Yang and U. Pal contributed equally to this work.

    Abbreviations used in this paper: Bb, Borrelia burgdorferi OspA, outer surface protein A OspB, outer surface protein B Strep, streptomycin Strepr, streptomycin resistance.

    Xiaofeng F. Yang, Utpal Pal, Sophie M. Alani, Erol Fikrig, Michael V. Norgard Essential Role for OspA/B in the Life Cycle of the Lyme Disease Spirochete . J Exp Med 1 March 2004 199 : 641648. doi:

    The molecular basis of how Borrelia burgdorferi , the Lyme disease spirochete, maintains itself in nature via a complex life cycle in ticks and mammals is poorly understood. Outer surface protein A of Bb has been the most intensively studied of all borrelial molecular constituents, and hence, much has been speculated about the potential role of OspA in the life cycle of Bb. However, the precise function of OspA heretofore has not been directly determined, due primarily to the inability to generate an OspA/B-deficient mutant from a virulent strain of Bb. In this study, we created an OspA/B-deficient mutant of an infectious human isolate of Bb and found that OspA/B function was not required for either Bb infection of mice or accompanying tissue pathology. However, OspA/B function was essential for Bb colonization of and survival within tick midguts, events crucial for sustaining Bb in its natural enzootic life cycle.

    Recommended Reading: Do Antibiotics Cure Lyme Disease

    View Live Lyme Disease Spirochetes Allegedly Easily Seen In Blood Following Negative Official Testing

    [CanLyme note: Although this video clearly shows a spirochaetal shaped bacteria, it cannot identify what spirochaete it may be. To show that those bacteria are in fact Borrelia , proper DNA sequencing would have to occur. From our perspective, if the person is sick and these are showing up in their blood, then treat the patient for a spirochaetosis while waiting for confirmation. Treatment is chosen within the patient/doctor discussion as to type of antibiotic and duration. Infectious Disease Society of America guidelines are not recommended.

    Current Canadian medical policy is to simply defer to the IDSA guidelines on matters of testing and treatment therefore we cannot recommend following Canadian medical policy in any province until patient experts opinion is given full and equal voice in the writing of Canadian medical policy relative to Lyme borreliosis.]

    Slide 5

    These video clips are from an experiment in which 11 friends provided a tiny amount of fingertip blood on a microscope slide.

    All donors were met through patient support groups and have chronic illness.

    8 of 11 have been ill for 20 years or longer.

    9 have been diagnosed with M.E. or Chronic Fatigue Syndrome

    10 of 11 had negative NHS tests for Lyme borreliosis one was not tested.

    9 had private tests that were positive.

    These eleven donors represent a total of 235 years of illness and 170years of lost productivity.

    View more slides from counsellingme.com

    Does It Help With Treatment

    Dark Field Microscopy Blood – Lyme Borellia Spirochetes in RBC – Needle Like Alive

    A test helps with treatment only if it swings the decision to do a certain treatment from the normal default treatment. I can find no evidence that this alters treatment. A Lyme tests known to produce false positives does authorize/justify the use of antibiotics in CFS patients. This is now the system works.

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