Researchers from the Medical University of Lubeck in Germany say that they have failed to replicate the tantalizing evidence from a study published two years ago that found Chlamydia in almost all the Alzheimer's brains examined. The German authors used PCR and immunocytochemistry in an attempt to detect either chlamydia DNA or antigens in tissue samples from 20 deceased AD patients. They found no evidence of the bacterium, thereby replicating similar negative results published by a group from the University of Washington last year.

Brian Balin of the Philadelphia College of Osteopathic Medicine, lead author of the report that had found chlamydia in 17 of 19 Alzheimer's brains (and only one of 19 controls), sticks by his original findings. "Unfortunately, the two studies that have been performed have assumed that techniques found successful for other tissue samples could be applied to brain samples that were formalin-fixed and paraffin-embedded," says Balin, whose study used frozen tissue. Because of this and a number of other methodological concerns (see below), Balin believes comparing these studies to his is like comparing apples to oranges.

"Our findings hold such great implications as to how inflammation in the AD brain may be triggered by infection with C pneumoniae that we must demand that studies to replicate and/or validate our first report should be performed with the rigor and comparable techniques that will provide data that can truly be compared and analyzed," says Balin.—Hakon Heimer

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  1. This letter is in response to the recent article in Journal of Clinical
    Microbiology (38[2]:881-882, 2000) entitled "Failure to detect Chlamydia pneumoniae in brain sections of Alzheimer's Disease Patients" by Gieffers
    et al. Since we were the first to report on this issue (Balin,
    et al., 1998
    ), I wanted to address how the present report differs quite
    substantially from that which we reported.

    My comments are outlined below:

    The recent report by Gieffers,
    et al.
    does not address our previous research findings using any of
    the same protocols with which we made our findings. This point is acknowledged
    by Gieffers et al. in their discussion of results. In fact, another report
    that recently appeared in the literature (Nochlin,
    et al., 1999
    ) used similar protocols as did Gieffers et al and also
    failed to detect C. pneumoniae in the Alzheimer's brain. In neither report
    did the investigators try to replicate our findings in the way that was
    clearly outlined in our 20-page report.

    Given that such a different technical approach was taken, we can only
    offer explanations as to the difficulties one encounters in using these
    different approaches. In fact, we attempted to maximize our own technical
    capabilities to address a very difficult question in identifying and localizing
    an intracellular organism in the Alzheimer brain. For this reason and because
    of the body of literature from hundreds of different Chlamydia studies that
    demonstrate a tremendous variability in diagnosing, identifying, and localizing
    Chlamydia in clinical specimens, we went to the extreme in our investigations
    by using a large variety of techniques on different clinical specimens.

    These techniques included: (A) both PCR and RT-PCR for genetic analysis
    of C. pneumoniae from FROZEN tissues , not formalin-fixed paraffin embedded
    tissues; (B) immunohistochemistry on formalin-fixed paraffin-embedded tissues
    of 7-10 micron thickness, not on sections as thin as 4 microns as outlined
    in the present report; also antigen retrieval and antibody dilutions from
    our report were more varied and we used some different antibodies than used
    in the present report (we have found that different aliquots of the anti-Chlamydial
    antibodies have to be carefully titrated to maximize the immunoreaction);
    (C) electron and immuno-electron microscopy to identify the organisms in
    cells and tissues; (D) culturing from AD human brain homogenates the Chlamydia
    organisms into human monocytes. In the latest reports, electron microscopy,
    immuno-electron microscopy, and culturing from brain tissues were not performed.

    We are confident that organisms containing genetic sequences and expressing
    C. pneumoniae-related antigens were present in the samples examined by our
    group. Our findings hold such great implications as to how inflammation
    in the AD brain may be triggered by infection with C. pneumoniae that we
    must demand that studies to replicate and/or validate our first report should
    be performed with the rigor and comparable techniques that will provide
    data that can truly be compared and analyzed. We cannot stop short of intensifying
    these studies and we must work to develop standard techniques that can be
    applied by laboratories analyzing clinical specimens. To fall short of this,
    as we believe that the present publications do, only further demonstrates
    the technical difficulties found in the Chlamydiology arena!

    The fundamental question is what role C. pneumoniae plays in the pathogenesis
    of Alzheimer's disease. Others should be compelled to address this question
    as well. We are sure that the original observations of C. pneumoniae in atherosclerosis
    were received skeptically, but this organism is now being fully investigated
    for its role in that disease. We hope that similar investigations will be
    forthcoming in AD as well because, as most objective scientists would agree,
    we are far from understanding the basis for neurodegenerative processes
    as they may relate to infectious agents such as Chlamydia, Borrelia, Herpes
    simplex virus type I, and HIV.

    The following additional paragraphs further discuss specifically why
    we believe that the techniques used in the two reports which could not detect
    C. pneumoniae in AD brains were not optimal for this determination. The tissues
    used in the present article for PCR analysis were formalin-fixed and paraffin-embedded
    tissue samples. In our PCR analysis, we used frozen tissue samples to minimize
    the difficulties encountered in PCR from fixed and embedded tissues. In
    particular, during extraction and re-hydration of paraffin-embedded fixed
    tissue, fragmentation of the bacterial DNA may prevent amplification of
    target sequences of > 400 bases so that nested PCR would not detect the
    organism and the sensitivity of the technique does not apply due to the
    starting material. In the present study, the authors state that "Successful
    DNA extraction was ensured, since the PCR protocol used was previously evaluated
    for vascular tissue and proven to be substantially more sensitive than cell
    culture." This assumption is not necessarily valid when applied to
    extraction of formalin-fixed paraffin-embedded brain tissues. The reasons
    would include: that the organism presence and total bacterial burden in
    brain tissues may vary quite extensively from those present in systemic
    vascular tissues (eg, tissue specific tropism by a strain variant), that
    the organism in brain tissues may have undergone forms of degradation compromising
    the status of its DNA, and the copy number of the genes of interest may
    be very low. In fact, in an article in J Clin Micro (36:1512-1517,
    1998
    ), in which Marchetti et al. evaluated PCR in the detection of another
    organism from formalin-fixed, paraffin-embedded tissues, the authors came
    to the conclusion that the efficacy of PCR strictly depends on several amplification
    parameters which include DNA concentration, target DNA size, and the repetitiveness
    of the amplified sequence. Furthermore, in the present study, extraction
    of DNA with phenol-chloroform was described as using standard protocols.
    I have two problems with this description. First, there is no reference
    given here as to which standard protocol was used (there are numerous and
    varied protocols that can be used); second, one cannot assume that the extraction
    protocol was sufficient to extract the target DNA from C. pneumoniae organisms,
    even though it apparently was sufficient for extracting eukaryotic DNA.

    In the present study, the investigators used PCR primer sets that have
    been shown to amplify C. pneumoniae genes in other types of clinical samples
    (i.e., coronary artery tissues). However, these primer sets were not comparable
    to what we used in our original study, and they may or may not work on C. pneumoniae in brain samples. The difficulties that many laboratories have
    had in the PCR analysis of Chlamydia in numerous clinical samples, anywhere
    from cardiovascular tissues, arthritis tissues, lung tissues, etc., indicates
    that no standard PCR methodology has been successfully developed to detect
    genetic material correlating to infection with Chlamydia pneumoniae. Given
    the recognition of these difficulties by the investigators in the discussion
    section of their present report, I find it curious as to why analysis of
    Alzheimer samples was not performed in a manner that would at least approach
    the protocol that we used in our original studies.

    With regard to the immunocytochemistry experiments on which the present
    study reports, we can make the following observations:

    A. Our report used 7-10 micron thick formalin-fixed paraffin-embedded
    sections for immunocytochemistry experiments, whereas the present report
    used 4 micron thick sections. In our experience with C. pneumoniae infection
    in the AD brain, we believe that it is best to use 7-10 micron thick sections
    from paraffin-embedded tissues to obtain sections that include C. pneumoniae
    inclusions within glial cells. Given the typical deparaffinization protocols
    and rehydration, we believe that very thin paraffin sections are more friable
    and cells that contain C. pneumoniae inclusions are even more suspect for
    maintenance of integrity. Our tissue culture data supports this latter contention,
    in that the cytoplasm and cytoplasmic vacuoles of C. pneumoniae-infected
    cells is easily extruded following cytospinning at very low speeds (500-800
     rpm).

    B. Our report used antigen retrieval prior to primary antibody incubations;
    this report does not mention antigen retrieval methods.

    C. The antibodies used in our report included one (RR-402, Washington
    Research Foundation) of which was used in the present report. We used dilutions
    of 1:50 - 1:250 for this antibody and obtained our best results with the
    dilutions at 1:50. Intriguingly, we also used the DAKO distributed antibody
    from the same source (Wash. Res. Found.), but needed to use this lot at
    1:5 dilution. We believe that different lots of the RR-402 antibody have
    different antibody concentrations, and presently, must titrate for each
    lot. This discrepancy may account for both positive and negative results
    depending on the lot used. Curiously, in a different report (in Neurology
    53:1888, 1999
    ) by individuals from the University of Washington (location
    of Washington Research Foundation), Department of Pathology and Pathobiology,
    this antibody was not even used in their study that also failed to detect
    C. pneumoniae in Alzheimer's brain tissues. We find their failure to use, basically,
    the antibody to be remarkable given the association of this group with the
    Washington Research Foundation. More intriguingly, this latter group of
    investigators also used methods of immunocytochemistry and PCR that were
    shown to be successful for detection of C. pneumoniae in atheromatous arterial
    tissues (Kuo
    et al., 1993
    ), similar to that used by Gieffers et al. in the present
    J Clin Micro report. I am addressing this issue here because in both reports
    by Gieffers et al. and Nochlin et al. (the Neurology report cited above),
    neither used methodology clearly outlined in our manuscript in Med. Micro.
    Immuno. 187:23-42, 1998.

    It is our belief that our thorough report using PCR, RT-PCR, immunocytochemistry,
    electron microscopy, immunoelectron microscopy, and culture analysis, along
    with all proper controls, should at least be mirrored in a comparable study
    to obtain results that could be realistically compared for their techniques
    and any discrepancies that may or may not be found. Unfortunately, the two
    studies that have been performed have assumed that techniques found successful
    for other tissue samples could be applied to brain samples that were formalin-fixed
    and paraffin-embedded. Therefore, in our estimation, these studies are comparing
    apples to oranges, and in essence they are just reaffirming the technical
    difficulties and absence of standardization of techniques that are used
    throughout the field of Chlamydiology and for application to clinical samples.

  2. Another organism linked to AD. Why has this not been seen before?

    View all comments by Paul Coleman

References

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Further Reading

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Primary Papers

  1. . Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain. Med Microbiol Immunol. 1998 Jun;187(1):23-42. PubMed.
  2. . Failure to detect Chlamydia pneumoniae in brain sections of Alzheimer's disease patients. J Clin Microbiol. 2000 Feb;38(2):881-2. PubMed.
  3. . Failure to detect Chlamydia pneumoniae in brain tissues of Alzheimer's disease. Neurology. 1999 Nov 10;53(8):1888. PubMed.