Dr Charles Forsyth

General Medical Practitioner

 
 

Medical microbiological investigations tend to fall into two main groups:

  1. Those that identify (and quantify) organisms or specific parts of organisms themselves.

  2. Those that measure the body’s response to organisms.

Both have their strengths and weaknesses.


Please remember that I am not a microbiologist, infectious disease specialist nor immunologist!  So I don’t have extensive experience in these areas - but am constantly learning and always keen to achieve a better understanding!



TESTS FOR THE ORGANISMS THEMSELVES


CULTURE

This is where the test samples are added to a culture medium which contains various nutrients (eg. a broth, blood,  carbohydrates, etc) known to be necessary for the growth of the organism being looked for.  The culture medium may be an agar gel in a petri dish or a liquid culture.  These are then incubated at a specific temperature known to be ideal for the organism being looked for.  The appearance of the colonies grown may then be identified by microscopy.  Culturing viruses requires a culture medium of living cells.  When organisms are successfully grown in culture they can then be tested for their sensitivity to specific antimicrobials to identify those agents that are most and least effective at killing them.


The success of culture is dependent on a number of factors, including: taking adequate samples; getting samples to the laboratory and processing them before too many of the organisms die; having a good idea of which organisms you are looking for, etc.



MICROSCOPY

This relies on visualizing and identifying organisms in samples collected, whether pus, sputum, urine, stool, blood, biopsies of affected tissues, etc - with or without culture.  Detection is dependent on the level of infection - the number of organisms present at the time the sample is taken.  A variety of different stains may be employed that bind to specific structures of the organism. 



DNA-PCR

This test directly detects and quantitatively measures DNA sequences specific to the organism being looked for.  It is therefore a measure of the amount of the organism present in the tissue being sampled.  It needs to be remembered that the distribution of organisms may vary greatly between different tissues, and organisms may be hidden in biofilm - so false-negatives are possible.  It can be carried out on blood and other fluids/tissues, eg. CSF, synovial fluid, biopsy samples, etc.  My impression is that in general it is not a very sensitive test, so if I am right, may yield ‘false negative’ results.



TESTS FOR THE BODY’S RESPONSE TO ORGANISMS


There are two main ways the immune system responds to infective organisms that we can measure:

  1. 1)Antibody production by B Lymphocytes - the ‘humoral immune response’, measured by ‘serological tests’.

  2. 2)Cell Mediated Immune Response. 

Both appear to be equally important - and the ideal is to test for both.

It is vital to remember that when there is immune suppression (whatever the cause) and quite often in chronic infections, either or both of these immune responses may be reduced - so that negative or mildly positive results may be mistakenly interpreted - what we call ‘false negative’ or relatively ‘false negative’ results.



SEROLOGY TESTS

These measure the amount of specific antibodies produced by B-cell lymphocytes to specific antigens of the organism being tested for. 

  1. IgM antibodies are usually produced early in the acute phase of an infection - so when IgM is positive it usually indicates active acute infection.  IgM antibodies may also be elevated when there is re-exposure to the infective organism or reactivation of a past infection with an organism.

  2. IgG antibodies typically appear weeks or months after initial infection and may continue to be present indefinitely.  They are generally considered to be indicative of an individual's immune status to the particular organism - in other words, evidence of previous infection.

    1. Positive IgG with negative IgM - is usually taken as evidence of past infection with no evidence of current active infection.  However if there is immune suppression, this may be misleading, and one should be more suspicious of this if the IgG levels are particularly high.  An EliSpot test (for cell mediated immunity) may reveal evidence of current active infection.

  3. IgA antibodies - these are rarely tested for in the UK (when testing for infective organisms), but is available via ArminLabs (Germany).  When IgA is positive, it usually indicates current active infection.


In my view, both IgM and IgG, and ideally IgA, should usually be tested in order to obtain a more complete picture.


Antibody production to a specific organism may be affected by a variety of factors which may yield false negative or false positive results, these include: other current infections, autoimmune disease, some medications, how hidden from the immune system the organism is (eg in biofilm), and for some organisms, their morphological form (eg. Lyme disease when the spirochaete transforms in to a cystic form). 


Tests for measuring antibodies include:

  1. Elisa (Enzyme-Linked Immunosorbent Assay, EIA) is a serology test that is aimed at the detection of the patient’s production of specific antibodies (IgG or IgM) to specific antigens of the organism.  However, some infections do not always result in a predictable antigen production and any test identifies only a small subset of antigenic proteins - which may sometimes result in false negatives.

  2. Western Blot - is a qualitative immunoassay of specific antibodies against specific antigen molecular weights of an organism.  It is used for some specific infections, including: HIV, Bovine spongiform encephalopathy (BSE), Lyme disease, Hepatitis B and HSV-2 (Herpes Type 2).

  3. SeraSpot - Armin Labs also offers the SeraSpot test for detecting Borrelia (Lyme) antibodies, this appears to be more specific and sensitive than the ELISA test.



Cell-Mediated Immunity tests

Cell mediated immunity is an immune response that does not involve antibodies, but instead includes:

  1. The activation of macrophages and natural killer T-lymphocytes

  2. The activation of antigen-specific cytotoxic T-lymphocytes

  3. The release of various cytokines in response to an antigen.

The cell medicated immune response is an absolutely vital response to infection, especially viral infections and  intracellular bacteria, and also plays important roles in defence against fungi, protozoans and cancers.


The main test currently used is:

  1. EliSpot (Enzyme-Linked Immunosorbent Spot Assay) LTT (Lymphocyte Transformation Test) measures the number of activated T-lymphocytes in cell cultures that release the cytokine Interferon Gamma after being challenged by a chosen specific microbial antigen.

  2. It is now a very well researched and well accepted test in general, however it is yet not accepted as valid in the UK - so it may be dismissed out of hand by the majority of UK doctors!! 

  3. It appears to be mainly used for Mycobacterium tuberculosis at present (and I think in the UK), and is being increasingly used worldwide for other infectious diseases, including Lyme disease, EBV, CMV, Herpes simplex (HSV1 & 2) and other Herpes viruses, Chlamydia pneumoniae, Chlamydia trachomatis, Mycoplasma pneumoniae, Ehrlichia/Anaplasma, Yersinia and others.

  4. EliSpot testing is not yet available for all the infective agents.



TESTS OF IMMUNE FUNCTION


I am not an immunologist, so don’t have great experience concerning this area - but am always keen to learn!


TOTAL IMMUNOGLOBULINS - IgA, IgG, IgM

  1. Low levels of one indicate reduced production (or an increased demand not being appropriately met) and are likely to reduce once’s ability to respond to infection.

  2. Elevated levels of one indicate increased production, and causes include a response to infection.

  3. Abnormal immunoglobulin levels should always be investigated further - there are many potential causes.



LYMPHOCYTE SUBSETS

I am not yet very knowledgable about this subject, however the following is what I currently understand (please correct me if I am wrong!)!

Lymphocytes are one of the five main types of white cells in the blood (the others being neutrophils, macrophages, monocytes and eosinophils). 

Lymphocytes are the main type of cell found in lymph and divide into two main types: B and T lymphocytes.

B lymphocytes provide humoral, antibody-driven adaptive immunity.

The are many sub-types of T lymphocytes, which include: Natural killer cells (NK cells) (for cell-mediated, cytotoxic innate immunity), Cytotoxic T cells (for cell-mediated, cytotoxic adaptive immunity), Helper T cells (send signals to other types of immune cells)


CD3 is a subset of T-cell lymphocyte that is necessary for activation of Cytotoxic T-cell lymphocytes.

  1. Low CD3 represents chronic immune-suppression, almost exclusively due to chronic viral infection.

  2. Elevated CD3 indicates immune response to viral infection, the most common being: EBV, CMV, HSV, VZV, Coxsackie.


CD4 T helper cells, also called CD4 cells, T-helper cells or T4 cells. They are called helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells, which then destroy the infectious particle.  When CD4 is low the body is left vulnerable to a wide range of infections that it would otherwise have been able to fight.

  1. Low CD4 represents immune suppression, eg. from an untreated infection (eg. HIV), or intentional immune suppression prior to transplant surgery.


CD8 relates mainly to cytotoxic T cells and natural killer (NK) T-cell lymphocytes.


CD57 is a subset of natural killer (NK) T-cell lymphocytes.  Low levels indicate immune suppression.  CD57 is down-regulated by the Th-1 cytokines (Interferon-gamma (IFNγ), Interleukin-2 (IL-2) and Tumor necrosis factor-alpha (TNF, TNFα).  Cloned T-cells from patients with chronic Lyme disease usually produce increased amounts of Th-1 cytokines.  A study from Dr Ray Stricker in 2001 showed low CD57 cells in nearly all chronic Lyme patients with Neuroborreliosis.

  1. Low CD57 represents immune suppression usually caused by chronic intracellular bacterial infection, most commonly Lyme Borrelia burgdorferi, Chlamydia pneumoniae, Mycoplasma pneumoniae. 


LYMPHOCYTE SUBSETS TESTS

  1. ArminLabs does a simple but very useful Lymphocyte Subset test which includes CD3, CD57 and NK cells (CD56) for £107.  I use this a lot, an d like to do it when ever I suspect multiple chronic infections.

  2. Lab4More does a much more comprehensive test “MicroImmunStatus”, but it is very complex and I have almost no experience with it yet.

  3. TDL do the following, but again I have very very little experience with them.

    1. Lymphocyte Subsets “LYSS” - CD3, CD4, CD8 - £196.

    2. Natural Killer Profile 1 “NKP1” - CD16, CD56, CD69 - £216

    3. Natural Killer Profile 2 “NKP2” - CD16, CD56, CD69 + CD3, CD4, CD8 - £216

    4. Natural Killer 1 “NK1” - CD16, CD19, CD56 - £523



RESOURCES

  1. Armin Labs  www.arminlabs.com

  2. TDL  https://tdlpathology.com

  3. Lab4More  www.lab4more.de

  4. Information sheets are available from the office (full list here) for patients:

  5. Infection Prevention

  6. Immune Support

  7. Infections Contributing to Chronic Diseases

  8. Inflammation

Microbiology Investigations

   INVESTIGATIONS: Nutritional  Gut  Toxins  Microbial  Mitochondrial  DNA Adducts