On 2018 Feb 04, Sin Hang Lee commented:

The medical profession, including medical schools and hospitals, is now a part of the health care industry, and implementation of editorial policies of medical journals is commonly biased in favor of business interests. PubMed Commons has offered the only, albeit constrained, open forum to air dissenting research and opinions in science-based language. Discontinuation of PubMed Commons will silence any questioning of the industry-sponsored promotional publications indexed in PubMed.

On 2017 Sep 25, Sin Hang Lee commented:

The research paper by Lager and colleagues [1] reported the first attempt to compare the sensitivity and specificity of any direct nucleic acid-based test protocols used by different laboratories in detecting the DNA of various borrelial species. Such efforts, if continued, may eventually lead to development of a useful direct test for reliable diagnosis of Lyme borreliosis (LB) and should be encouraged worldwide.

The authors’ statement “PCR is not suitable as a primary diagnostic tool for Lyme borreliosis (LB)” may be too dogmatic. If a PCR test can be proven to be sensitive and specific for reliable diagnosis of LB, there is no reason to reject it as a primary diagnostic tool since several co-authors of this article have stated in a recent position paper that clinicians are advised to avoid serological testing whenever the clinical symptoms are not indicative of LB according to the case definitions because the current serology tests are of little diagnostic value. [2]

It is no surprise that the authors found that the16S rRNA PCR protocols have a higher analytical sensitivity than the non-16S rRNA PCR protocols and the concordance between the 16S rRNA PCR protocols is high. The bacterial 16S rRNA gene is species-specific, is essential for protein synthesis and does not mutate in our lifetime. Non-16S rRNA genes in a borrelia strain may mutate or be deleted, and may be shared by other species of bacteria.

However, using real-time PCR to detect a signature segment of borrelial 16S rRNA gene for the diagnosis of Lyme borreliosis is bound to encounter technical difficulties. For clinical diagnostic real-time PCR, the optimal amplicon length is usually less than 150 bp [3], often below 80 bp [4], as illustrated in this article. [1] Since all bacterial species have a 16S rRNA gene consisting of about 1500 bp of DNA with interspecies highly conserved segments and hypervariable segments of sequence intercalated between themselves, PCR primer selections are crucial. The false positive B. hermsii and B. miyamotoi detection in protocols 3, 7 and 8 illustrates the inevitable errors of depending on probe hybridization to distinguish closely related DNA sequences. The authors have finally reached a correct conclusion that “In practice, all 16S PCR-based tests without DNA sequencing of the PCR amplicon for validation are prone to generate this kind of error”. In order to detect Borrelia spielmanii, Borrelia lusitaniae and Borrelia japonica, a pair of more inclusive genus-specific PCR primers [5] may be needed.

The recommendation “to complement the real-time PCR results with sequencing results” is not practical for the diagnosis of Lyme borreliosis. The amplicon generated by real-time PCR primers cannot be used for automated Sanger sequencing because the fragments are too short. If the real-time PCR technology were used to screen patient materials like the blood samples which contain human genomic DNA, there would be many questionable positive results which need to be re-tested by conventional PCR to prepare suitable templates for Sanger reaction.

In their research as reported [1], the authors distributed cDNA and extracted DNA in water or buffer solution from pure borrelial cultures and normal cerebrospinal fluid spiked with pure borrelial cultures as the testing materials which do not contain human genomic DNA as interfering substances. These experimental designs do not reflect the real conditions the diagnostic laboratories are facing.

It is recommended that a similar comparative research be conducted by sending out blind-coded EDTA blood samples spiked with different concentrations of various borrelial cultures to different laboratories for a proficiency test survey. There may be more appropriate direct tests out there which are better than real-time PCR for a reliable diagnosis of Lyme borreliosis.

References

[1] Lager M, Faller M, Wilhelmsson P, Kjelland V, Andreassen Å, Dargis R, Quarsten H, Dessau R, Fingerle V, Margos G, Noraas S, Ornstein K, Petersson AC, Matussek A, Lindgren PE, Henningsson AJ. Molecular detection of Borrelia burgdorferi sensu lato - An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories. PLoS One. 2017 Sep 22;12(9):e0185434.

[2] Dessau RB, van Dam AP, Fingerle V, Gray J, Hovius J, Hunfeld KP, Jaulhac B, Kahl O, Kristoferitsch W, Lindgren PE, Markowicz M, Mavin S, Ornstein K, Rupprecht T, Stanek G, Strle F. To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis. Clin Microbiol Infect. 2017 Sep 5. pii: S1198-743X(17)30488-3.

[3] Ornstein K, Barbour AG. A reverse transcriptase-polymerase chain reaction assay of Borrelia burgdorferi 16S rRNA for highly sensitive quantification of pathogen load in a vector. Vector borne and zoonotic diseases. 2006; 6:103-12.

[4] Tsao JI, et al. An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle. Proc Natl Acad Sci U S A. 2004 ;101:18159-64.

[5] Lee SH, et al. DNA sequencing diagnosis of off-season spirochetemia with low bacterial density in Borrelia burgdorferi and Borrelia miyamotoi infections. Int J Mol Sci. 2014;15:11364-86.

Conflicts of Interest: Sin Hang Lee, MD is the director of Milford Molecular Diagnostics Laboratory specialized in developing DNA sequencing-based diagnostic tests.

On 2017 Sep 25, Sin Hang Lee commented:

The research paper by Lager and colleagues [1] reported the first attempt to compare the sensitivity and specificity of any direct nucleic acid-based test protocols used by different laboratories in detecting the DNA of various borrelial species. Such efforts, if continued, may eventually lead to development of a useful direct test for reliable diagnosis of Lyme borreliosis (LB) and should be encouraged worldwide.

The authors’ statement “PCR is not suitable as a primary diagnostic tool for Lyme borreliosis (LB)” may be too dogmatic. If a PCR test can be proven to be sensitive and specific for reliable diagnosis of LB, there is no reason to reject it as a primary diagnostic tool since several co-authors of this article have stated in a recent position paper that clinicians are advised to avoid serological testing whenever the clinical symptoms are not indicative of LB according to the case definitions because the current serology tests are of little diagnostic value. [2]

It is no surprise that the authors found that the16S rRNA PCR protocols have a higher analytical sensitivity than the non-16S rRNA PCR protocols and the concordance between the 16S rRNA PCR protocols is high. The bacterial 16S rRNA gene is species-specific, is essential for protein synthesis and does not mutate in our lifetime. Non-16S rRNA genes in a borrelia strain may mutate or be deleted, and may be shared by other species of bacteria.

However, using real-time PCR to detect a signature segment of borrelial 16S rRNA gene for the diagnosis of Lyme borreliosis is bound to encounter technical difficulties. For clinical diagnostic real-time PCR, the optimal amplicon length is usually less than 150 bp [3], often below 80 bp [4], as illustrated in this article. [1] Since all bacterial species have a 16S rRNA gene consisting of about 1500 bp of DNA with interspecies highly conserved segments and hypervariable segments of sequence intercalated between themselves, PCR primer selections are crucial. The false positive B. hermsii and B. miyamotoi detection in protocols 3, 7 and 8 illustrates the inevitable errors of depending on probe hybridization to distinguish closely related DNA sequences. The authors have finally reached a correct conclusion that “In practice, all 16S PCR-based tests without DNA sequencing of the PCR amplicon for validation are prone to generate this kind of error”. In order to detect Borrelia spielmanii, Borrelia lusitaniae and Borrelia japonica, a pair of more inclusive genus-specific PCR primers [5] may be needed.

The recommendation “to complement the real-time PCR results with sequencing results” is not practical for the diagnosis of Lyme borreliosis. The amplicon generated by real-time PCR primers cannot be used for automated Sanger sequencing because the fragments are too short. If the real-time PCR technology were used to screen patient materials like the blood samples which contain human genomic DNA, there would be many questionable positive results which need to be re-tested by conventional PCR to prepare suitable templates for Sanger reaction.

In their research as reported [1], the authors distributed cDNA and extracted DNA in water or buffer solution from pure borrelial cultures and normal cerebrospinal fluid spiked with pure borrelial cultures as the testing materials which do not contain human genomic DNA as interfering substances. These experimental designs do not reflect the real conditions the diagnostic laboratories are facing.

It is recommended that a similar comparative research be conducted by sending out blind-coded EDTA blood samples spiked with different concentrations of various borrelial cultures to different laboratories for a proficiency test survey. There may be more appropriate direct tests out there which are better than real-time PCR for a reliable diagnosis of Lyme borreliosis.

References

[1] Lager M, Faller M, Wilhelmsson P, Kjelland V, Andreassen Å, Dargis R, Quarsten H, Dessau R, Fingerle V, Margos G, Noraas S, Ornstein K, Petersson AC, Matussek A, Lindgren PE, Henningsson AJ. Molecular detection of Borrelia burgdorferi sensu lato - An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories. PLoS One. 2017 Sep 22;12(9):e0185434.

[2] Dessau RB, van Dam AP, Fingerle V, Gray J, Hovius J, Hunfeld KP, Jaulhac B, Kahl O, Kristoferitsch W, Lindgren PE, Markowicz M, Mavin S, Ornstein K, Rupprecht T, Stanek G, Strle F. To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis. Clin Microbiol Infect. 2017 Sep 5. pii: S1198-743X(17)30488-3.

[3] Ornstein K, Barbour AG. A reverse transcriptase-polymerase chain reaction assay of Borrelia burgdorferi 16S rRNA for highly sensitive quantification of pathogen load in a vector. Vector borne and zoonotic diseases. 2006; 6:103-12.

[4] Tsao JI, et al. An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle. Proc Natl Acad Sci U S A. 2004 ;101:18159-64.

[5] Lee SH, et al. DNA sequencing diagnosis of off-season spirochetemia with low bacterial density in Borrelia burgdorferi and Borrelia miyamotoi infections. Int J Mol Sci. 2014;15:11364-86.

Conflicts of Interest: Sin Hang Lee, MD is the director of Milford Molecular Diagnostics Laboratory specialized in developing DNA sequencing-based diagnostic tests.

On 2018 Feb 04, Sin Hang Lee commented:

The medical profession, including medical schools and hospitals, is now a part of the health care industry, and implementation of editorial policies of medical journals is commonly biased in favor of business interests. PubMed Commons has offered the only, albeit constrained, open forum to air dissenting research and opinions in science-based language. Discontinuation of PubMed Commons will silence any questioning of the industry-sponsored promotional publications indexed in PubMed.