Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
Post Reply
Martian
Posts: 1944
Joined: Thu 26 Jul 2007 18:29
Location: Friesland, the Netherlands

Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by Martian » Fri 1 Jul 2016 17:59

The study below checked the LM-method. I think LM stands for Laane and Mysterud, who developed and published about this microscopy method for detection of Borrelia and Babesia.

PubMed abstract:

Source: http://www.ncbi.nlm.nih.gov/pubmed/27030913
Infect Dis (Lond). 2016 Jun;48(6):411-9. doi: 10.3109/23744235.2016.1144931. Epub 2016 Feb 23.

Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood.

Aase A, Hajdusek O, Øines Ø, Quarsten H, Wilhelmsson P, Herstad TK, Kjelland V, Sima R, Jalovecka M, Lindgren PE, Aaberge IS.

Abstract

Background

A modified microscopy protocol (the LM-method) was used to demonstrate what was interpreted as Borrelia spirochetes and later also Babesia sp., in peripheral blood from patients. The method gained much publicity, but was not validated prior to publication, which became the purpose of this study using appropriate scientific methodology, including a control group.

Methods

Blood from 21 patients previously interpreted as positive for Borrelia and/or Babesia infection by the LM-method and 41 healthy controls without known history of tick bite were collected, blinded and analysed for these pathogens by microscopy in two laboratories by the LM-method and conventional method, respectively, by PCR methods in five laboratories and by serology in one laboratory.

Results

Microscopy by the LM-method identified structures claimed to be Borrelia- and/or Babesia in 66% of the blood samples of the patient group and in 85% in the healthy control group. Microscopy by the conventional method for Babesia only did not identify Babesia in any samples. PCR analysis detected Borrelia DNA in one sample of the patient group and in eight samples of the control group; whereas Babesia DNA was not detected in any of the blood samples using molecular methods.

Conclusions

The structures interpreted as Borrelia and Babesia by the LM-method could not be verified by PCR. The method was, thus, falsified. This study underlines the importance of doing proper test validation before new or modified assays are introduced.

KEYWORDS:

Babesia spp.; Borrelia burgdorferi sensu lato; Lyme borreliosis; Lyme disease; PCR; babesiosis; microscopy

PMID: 27030913
DOI: 10.3109/23744235.2016.1144931

[PubMed - in process]
Original source and free full text: http://www.tandfonline.com/doi/full/10. ... 16.1144931

X-member
Posts: 6002
Joined: Mon 30 Jul 2007 18:18

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by X-member » Fri 1 Jul 2016 18:20

Prof Morten M. Laanes paper is out - detection method

http://www.lymeneteurope.org/forum/view ... e&start=60

A quote (Sun Feb 28, 2016):
I have today been informed that professor Laane and Mysterud forbade FHI (Institute of Public Health) to use their names because FHI went out in advance with the results before L + M was able to explain what they actually meant, which was a violation of the contract.

Martian
Posts: 1944
Joined: Thu 26 Jul 2007 18:29
Location: Friesland, the Netherlands

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by Martian » Fri 1 Jul 2016 18:22

Source: http://www.alphagalileo.org/ViewItem.as ... ureCode=en
News Release

Researchers uncover potential flaws in test for Lyme Disease

30 June 2016, Taylor & Francis

A new microscopy technique (LM-method) developed to detect Lyme disease is unable to distinguish infected patients from healthy controls, yielding false-positive results that could lead doctors to over-diagnose a patient, according to new research published in the journal Infectious Diseases.

The new research follows up on a previous study suggesting that modified microscopy techniques (LM-method) could detect active cases of Lyme disease (caused by Borrelia bacteria) and Babesia (a tick-borne malaria-like parasite) in just one to two days. Despite considerable publicity and patient demand for this test in Norway, earlier studies did not include a control group and methods were not validated and ready for use in patients.

To investigate the reliability of the new test, Dr Audun Aase, from the Norwegian Institute of Public Health, and his colleagues collected blood samples both from people who had been suffering from Lyme disease-like symptoms for several years and previously tested positive for Borrelia and/or Babesia by the LM-method (21 people), and healthy controls with no known history of tick bites (41 people). The samples were then masked and analyzed in independent laboratories using a range of diagnostic tests including the LM-method, conventional microscopy, genetic fingerprint testing (PCR), and serology.

The results indicate that the new LM-method can trigger false positives, suggesting people have Lyme disease when they really don’t. Using the LM-method, 14 (66%) patient group blood samples and 35 (85%) control group samples were judged positive for Borrelia and/or Babesia. However, only one sample (5%) of the patient group and eight samples (20%) of the control group tested positive for Borrelia DNA by PCR. None of the samples were positive for Babesia DNA, and conventional microscopy did not identify Babesia in any of the samples.

In an accompanying editorial commentary, Dr Ram B. Dessau, an expert on infectious diseases and Senior Consultant at Slagelse Hospital, Slagelse, Denmark, writes, “I hope the study serves as a warning against non-validated microscopic procedures and helps prevent mismanagement of patients with chronic complaints, who are lured to seek improper diagnosis in the future.”

Lyme disease is the most common tick-borne infection in Europe and North America, with 360,000 cases reported over the last 20 years in Europe alone. While most people who contract the Lyme disease recover quickly after antibiotic treatment, up to a fifth of patients report persistent symptoms years after they have been told standard tests are negative for the disease. Interest in new diagnostic tests is therefore high.

Martian
Posts: 1944
Joined: Thu 26 Jul 2007 18:29
Location: Friesland, the Netherlands

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by Martian » Sat 2 Jul 2016 0:31

Source and free full text: http://www.tandfonline.com/doi/full/10. ... 16.1144932
Infectious Diseases
Volume 48, Issue 6, 2016

Editorial Commentary

Microscopy of human blood for Borrelia burgdorferi and Babesia without clinical or scientific rationale

DOI: 10.3109/23744235.2016.1144932

Ram B. Dessau
pages 420-421

Received: 11 Jan 2016
Accepted: 18 Jan 2016
Published online: 17 Feb 2016

In Norway, ‘A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques’ has attracted public discussion and media attention.[1] This method of ‘simple microscopy’ was developed by Mysterud and Laane from the department of Molecular Biosciences and Biology at Oslo University. A validation study has now been performed and published in the current issue of Infectious Diseases. The ‘simple microscopy’ was assessed thoroughly and found to be useless.[2] Masked samples from cases previously judged positive by the ‘simple microscopy’ and from a healthy control group were included. Using the ‘simple microscopy’, more than 50% of cases and more than 80% of controls were judged positive. PCR performed by independent laboratories did not detect Borrelia or Babesia DNA. Babesia was also not found by conventional microscopy.

In any case microscopy of blood is not a relevant procedure for the diagnosis of Lyme borreliosis for the following reason. Although haematogenous dissemination has been demonstrated by PCR and culture, the concentration of B. burgdorferi has been estimated to be about one bacterial cell in 10 ml of blood in patients with erythema migrans.[3,4] Thus, the concentration of Borrelia in patients has been shown to be too low to be found in a droplet of 6 μl on a microscope slide. A 10000-times higher concentration would be needed to achieve a reasonable sensitivity by microscopy.

‘Simple microscopy’ is not a relevant procedure for Babesia either, as conventional staining of the blood film is necessary for visualisation and correct identification.[5]

It would be unlikely that patients assumed to have concentrations of 1000 or more microorganisms per millilitre of blood have heterogeneous chronic symptoms for years without developing more severe pathology. By comparison, patients with high concentrations of microorganisms, as is usually seen with Babesia or relapsing fever Borreliae, are described as febrile and present with severe acute disease.[4,5]

The problem of misinterpretation and poor preparation of microscopic slides is historic. Koch,[6] in a publication from 1877, was very particular about methods for microscopy. Koch warns the reader of a swelling dirty stream [‘… zu einem trüben Strohm anschwellen lassen…’] of publications on bacteriology. Koch stressed the importance of preserving the intact morphology of the microorganisms.

B. burgdorferi, in particular, has become the victim of a swelling dirty stream of problematic publications. In 2010 an independent review committee of the Infectious Diseases Society of America assessed 1025 publications and found a large volume of uncontrolled observations of doubtful scientific quality.[7,8]

The background for the study by Laane and Mysterud [1] is a speculative theory that morphological variants of microorganisms play a role in otherwise unexplained persisting symptoms. The literature does not support any causal relationship.[9]

Light microscopy is an important basic procedure in microbiology using wet mounts and stained preparations. How to perform microscopy is in the basic curriculum for undergraduate students in microbiology and for laboratory technicians. It is part of basic training to identify microorganisms and to distinguish tissue structures or artefacts.

It is reprehensible that pseudoscientific diagnostic testing was performed on human specimens at Oslo University. This practice has now been stopped, to my knowledge. However, non-validated diagnostic procedures are still be provided by alternative private laboratories and clinics and this has been warned against previously.[10–12]

If patients do not suffer from Lyme borreliosis or another tick-borne infection, then what is the problem?

Experience from a Dutch infectious disease clinic specialising in suspected Lyme borreliosis found 18 (9%) of 200 consecutive cases to have definite or probable Borrelia infection.[13] Notably, no alternative diagnosis was found in 77 (39%), despite a multidisciplinary approach. Probably most of the patients seeking alternative medical attention for a perceived tick-borne disease are recruited from the latter group without an ‘alternative diagnosis’. This labelling illustrates the problem, as the patient may feel rejected and lose trust. The patients most likely suffer from a somatoform disorder and require psychological and social support.[14] The psychological attention should start concurrent with the standard clinical work-up, not at a later stage, in order stop to progression of somatoform disorders. It is also recommended to avoid unnecessary diagnostic testing.[15] Borrelia serology should be avoided in patients with diffuse symptoms without objective designs of disease.[16] The positive predictive values will be low (<1%) and a positive serology result will only contribute to unnecessary concern.

In conclusion, Aase et al. [2] should be recommended for their effort to falsify inappropriate microscopy. The study shows the fundamental importance of a control group for evaluation of diagnostic accuracy and that cases with disease should be selected according to appropriate case definitions. I hope the study serves as a warning against non-validated microscopic procedures and helps prevent mismanagement of patients with chronic complaints, who are lured to seek improper diagnosis in the future.

Ram B. Dessau

Region Sjælland, Clinical Microbiology, Ingemannsvej 18 Slagelse, DK4200, Denmark
ramd@regionsjaelland.dk
See source for references.

Martian
Posts: 1944
Joined: Thu 26 Jul 2007 18:29
Location: Friesland, the Netherlands

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by Martian » Sat 2 Jul 2016 1:06

For more information and discussion about the research article titled "A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques" by Morten M. Laane and Ivar Mysterud, published in Biological and Biomedical Reports (2013), see LNE topic Prof Morten M. Laanes paper is out - detection method (6 Mar 2013).

User avatar
inmacdonald
Posts: 977
Joined: Fri 13 Jan 2012 22:32

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by inmacdonald » Mon 4 Jul 2016 16:56

PCR ( polymerase Chain Reaction amplification of specific target DNA)
is , within PCR LIMITS , a contributory technology for investigations seeking
infectious microbes in clinical specimens.

Eisendle and colleagues, have published
in their "Focus Floating Microscopy..." peer reviewed publication in the
American Journal of Clinical Pathology in year 2007... This is an excellent
side by side comparison of the Sensitivity and Specificity of PCR techniques
in multiple cases of Microscopically Immunohistochemistry CONFIRMED
Borrelia Human Infections.

PCR results , when matched against Focus Floating Microscopy ( Immunohistochemistry)[ IHC]
FAILED TO RATIFY BORRELIA INFECTIONS IN HUMAN SKIN in approximately ONE HALF of the
Microscopy POSITIVE Cases.



Conclusion:

-PCR is declarative in only about ONE HALF of Microscopy POSITIVE for borrelia detections ( FFM method)

Only POSITIVE results count in IMMUNOPATHOLOGY

- Immunohistochemistry results in Eisendle's patient Study of Borrelia in human skin produced
TRUE POSITIVE RESULTS _ ___IHC stains the Borrelia in skin
and
FALSE Negative RESULTS ____PCR returns only 50% Positives in the Microscopic Positive Matched cases

Respectfully,
Alan B. MacDonald, MD, FACP
July 4, 2016

User avatar
inmacdonald
Posts: 977
Joined: Fri 13 Jan 2012 22:32

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babes

Post by inmacdonald » Mon 4 Jul 2016 20:16

Our Webmaster now , today, July 4, 2016, learned that PCR
DOES NOT TRUMP
MICROSCOPIC DIAGNOSES OF BORRELIA--
Ever!
He now apologizes to his readership on this forum..
By

HIS POST OF a new subject entitled
Focus Floating Mucroscopy: 'Gold Standard' for cutaneous Borreliosis
THIS FROM YEAR 2007..
Http://ajcp.oxfordjournals.org/content/127/2/213.long

-------------

The Eisendle article and Abstract and Excerpt
, separately posted on July 4 2016 by Martian ( Above)-
...?coincidences???

Clearly prove the Triumph of Microscopy over
Mere PCR evidence..
These Facts contained in Eisendle et al: PCR MISSES more than 50% of patients , ticks, tick bites,skin biopsies
( total cases examined - 263)
who, under the Microscope
With Focus Floating Microscopy who are POSITIVE For BORRELIA INFECTIONS IN THEIR SKIN BIOPSIES

American Journal of Clinical Pathology ,Focus Floating Microscopy .. From 2007 Eisendle et al
AND....Previously posted by Inmacdonald,On thIS VERY Subject

----
MacDonald Conclusion:

PCR method misses 50% of Biopsy positive Borrelia infections!!!!!!!!!

Shock and Awe!,
How can this possibly be?

Answer:
The PCR TEST USED PCR PRIMERS WHICH WERE NOT DESIGNED TO RECOGNIZE
ALL SPECIES OF BORRELIA
---
Therefore a negative result in PCR,...
Never excludes the possibility ....,
That the Borrelia.....,Under the microscope...,Is actually not B31 strain Borrelia l...
I.e.......
Miyamotoi Borrelia
Or
Mayonii Borrelia
Or
An Australian Borrelia
Or
Borrelia bissetti
Or
Borrelia Valaisainia
Or,__________
Or___________
Or____________

Fill in the blanks on this page
And
Be aware of the BLANKS IN THE KNOWLEDGE IN THE MINDS OF THOSE
WHO SPURN THE INTRINSIC SUPERIORITIES OF
PROOFS MICROSCOPIC,,!!!!
Over
Mere negative PCR results

Respectfully
Alan B. MacDonald MD, FCAP
JULY 4, 2016

User avatar
ChronicLyme19
Posts: 564
Joined: Mon 11 Aug 2014 17:42
Location: NY, USA

Re: Validate or falsify: Lessons learned from microscopy method claimed to be useful for detecting Borrelia and Babesia

Post by ChronicLyme19 » Sun 10 Jul 2016 21:03

Thank you Dr. MacDonald for the explanation.
Half of what you are taught is incorrect, but which half? What if there's another half missing?

Post Reply