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Patient and doctor comments on PCR paper

The December, 1996 edition of the Journal of Clinical Microbiology includes an article by Dr. John Krieger and his team at the University of Washington on the potential for use of PCR, an advanced molecular biology technique, in diagnosing prostatitis cases which do not reveal any bacteriological cause by more conventional means.
What follows on the remainder of this page is a collection of patient comments on the PCR paper collected from the newsgroup sci.med.prostate.prostatitis .

From: Brad Hennenfent, MD

December 16, 1996

The Study by John Krieger, MD is a landmark in urology and is the most important study of the prostate ever done in this reviewers opinion. I would like to point out what it seems to say:

  1. All non-bacterial prostatitis is probably bacterial. Using meticulous controls for skin contamination, the study found that 77% of "non-bacterial" prostatitis patients as defined by the now antique (25 year old) Meares and Stamey 4 culture test, have bacterial DNA in their prostate biopsy specimens when they are tested for bacterial DNA using the polymerase chain reaction (PCR).

  2. An addtional 10% of the 135 patients had Mycoplasma, Chlamydia, or Trichomonas. Adding this to the 77% with bacterial DNA means that 87% of the patients with so called non-bacterial prostatitis had microorganisms in their prostates.

  3. The 77% that had bacteria had bacteria that have never before been cultured or discovered. Apparently, two or more new species of bacteria have been found that are related to prostatitis. What antibiotic would work in these cases is totally up in the air. Krieger's group, I assume, ran out of money to keep sequencing the DNA that they found and thus only sequenced a small proportion of the bacterial DNA that they found.

  4. Now all this DNA from the study needs to be sequenced and methods to culture these unculturable organisms need to be developed. Once this is achieved, antibiotic sensitivities can be determined.

  5. Since prostatitis was found in 97% of cases of BPH in one study, and is always found in over 50% of prostates being biopsied to rule out cancer, these DNA techniques should immediately be used on those patients as well.

  6. Krieger and his University of Washington group have clearly emerged head and shoulders above everyone else in the USA at this time as leaders in prostatitis research.

Journal of Clinical Microbiology
Volume 34, Number 12. December 1996
Prokaryotic DNA Sequences in Patients with Chronic Idiopathic Prostatitis
John N. Krieger, Donald E. Riley, Marilyn C. Roberts, and Richard E. Berger 3120-3128

IMHO, this is a landmark paper for our disease. Accordingly, I am copying the abstract for all to read :

"Half of all men experience symptoms of prostatitis at some time in their lives, but the etiology is unknown for more than 90% of patients. Optimal clinical and culture methods were used to select 135 men with chronic prostatitis refractory to multiple previous courses of antimicrobial therapy. The subjects had no evidence of structural or functional lower genitourinary tract abnormalities, of bacteriuria or bacterial prostatitis by traditional clinical tests, or of urethritis or urethral pathogens by culture. Specific PCR assays detected Mycoplasma genitalium, Chlamydia trachomatis, or Trichomonas vaginalis in 10 patients (8%). Broad-spectrum PCR tests detected tetracycline resistance-encoding genes, tetM-tetO-tetS, in 25% of patients and 16S rRNA in 77% of subjects. The tetM-tetO-tetS-positive cases constituted a subset of the 16S rRNA-positive cases. Patients with 16S rRNA were more likely to have 1,000 (or more) leukocytes per cubic mm in their expressed prostatic secretions than men whose prostate biopsy specimens were negative for 16S rRNA (P less than 0.001). Based on direct sequencing and repetitive cloning, multiple sources of 16S rRNA were observed in individual patients. Sequences of 29 cloned PCR products revealed 16S rRNAs distinct from those of common skin and gut flora.

These findings suggest that the prostate can harbor microorganisms that are not detectable by traditional approaches . [emphasis and set-aside mine]

These organisms may be associated with inflammation in the expressed prostatic secretions. Molecular methods hold great promise for identifying culture-resistant microorganisms in patients with chronic prostatitis."

This is very detailed, very careful work that has been in progress for 5 years. The patients selected for the study "passed" the Meares-Stamey culture procedure (or something like it) - no infective agent was found. The 135 patients selected for study underwent prostatic biopsy by a perineal approach (not through the rectum). The biopsy needle was introduced through a larger needle that penetrated the skin first. In this way, contamination from skin flora was avoided or minimized.

The PCR (polymerase chain reaction) studies were done by specialized laboratories with scrupulous anti-contamination measures. The DNA analyses were very sophisticated indeed.

The mean age of the subjects was 38.3.

In control experiments, it was demonstrated that the PCR method is more sensitive than culture in detecting C. trachomatis. For T. vaginalis, this sensitivity advantage of PCR over cultures was shown to be at least a factor of 10,000. For C trachomatis, T. vaginalis, herpes simplex virus, cytomegalovirus, M. genitalium, and U. urealyticum, it was shown that less than 10 organisms or "tissue-culture infecting units" could be detected.

10 patients were positive for one or more of the specific organisms named above. 5 had M. genitalium, 4 had C. trachomatis, and 2 had T. vaginalis. One had both M. genitalium and C. trachomatis. No patient was positive for genital viruses.

16S rRNA was positive for 103 or 134 patients, tetM-tetO-tetS was positive for 30 of 120 patients. Of these 30, 29 were also positive for 16S rRNA.

Sequencing of the 16S rRNA-encoding DNA was done partially or completely for 29 clones from 10 patients. Comparisons with a gene bank indicated the possible species involved: Vibrio furnissii, Staphylococcus aureus, Staphylococcus epidermidis, Escheria coli, and Aeromonas allosaccharophilia. To me, it is especially noteworthy to see the Staphs appearing in this list.

Patients typically exhibited more than one kind of 16S rRNA, implying infection from more than one kind of organism.

"[These findings are] not sufficient to establish that bacteria cause [chronic prostatitis]...Establishing a role for bacteria in the etiology of chronic prostatitis will likely involve multiple steps...Our DNA sequence data strongly suggest that the 16S rRNA PCR positives were from a source separate from known gut, skin, and laboratory contaminants. The correlation between 16S rRNA-positive and tetM-tetO-tetS-positive results is also consistent with the clinical observation that antibiotic therapy provides transitory, if any, relief of symptoms for many patients."

I intepret the last sentence to be a comment on the drug-resistance of the bacteria being detected, that is, they are saying that their finding of these genes for tetracycline-resistance in bacteria explains why antibiotic therapy is not successful or only transiently successful.

I'm thrilled with this work, but I'm not sure that anybody wants to wait until all the proof is in to be cured.

So chronic patients may harbor tetracycline-resistant bacteria? Let us assume so. Then what are these bacteria susceptible to? Is there an empirical regimen that can be recommended? How does all of this tie into drainage during therapy?

John Garst

PCR is an important bio-technology which uses a thermostable enzyme to faithfully reproduce fragments of DNA. The framents are doubled and doubled and doubled again until they have been copied thousands of times. This makes the sample being studied have enough copies of a piece of DNA so that it is easy to sequence it. The usefulness of this is to find out whether a particular sequence of DNA is present in a sample of living or once-living material. If one resorts to the needle-in-a-haystack metaphor, it reproduces the needle over and over again, so that it's fairly easy to look at the haystack and tell that there are needles in it and exactly what kind of steel the needles are made of and how long they are and whether they are used for fine thread or yarn, etc. A drop of blood or other bodily fluid contains billions of segments of DNA, but if even one of them is the segement you are looking for, you can show that it is present. An invaluable tool only barely being used for its potential in clinical diagnosis....

-- Ken Smith

Kreiger, Riley, Robers, and Berger, J Clin Microbiol 34, 3120-3128 (1996) (December issue), provide some interesting background for their work.

In 1980, it was estimated that "there were 20 office visits per 1,000 men per year for symptoms capatible with prostatitis."

[The following calculations are mine. At that rate, using the 1992 census figure of 87,078,000 men aged at least 20, this figures out to an estimated total of 1,741,560 office visits for "symptoms compatible with prostatitis" in the year 1992. If the average fee for these office visits and associated laboratory work was $75, then the cost to patients and their insurance companies was $130,617,000. Actually, I estimate the lifetime cost to society of my own case as over $300,000. If the number of men born each year who will develop cases like mine is 1 per 1,000, and if the birth rate is 2,000,000 males/year in the US, then each year the U.S. incurs a lifetime burden of 2,000 x $300,000 = $600,000,000. If this is spread evenly over 60 years, that is a cost of $5,000 per year per man. Applying that rate (along with the rate of 1 per 1000 being like me) to the 1992 census for men 20 and over, the annual cost to society is estimated as $435,390,000. It would not surprise me if the actual annual cost of prostatitis in the US were of the order of $1,000,000,000 (a billion dollars). Of course, we don't really get excited nowadays unless a cost is in the trillions, but still, a billion dollar is significant, I think.]

Kreiger et al. suspect that "chronic idiopathic prostatitis" (nonbacterial prostatits and prostatodynia) could be, in fact, bacterial. Part of the evidence is (a) that the onset of the disease is often related to urinary tract infections, (b) that "antimicrobial treatmentment is often transiently effective in relieving symptoms," and (c) that some (but not all) investigators have reported evidence for various pathogenic organisms. Few have evaluated prostatic tissue (instead of expressed fluids).

There are many problems with cultures of urine, semen, or expressed prostatic secretions: (a) inhibitory substances, (b) effects of many previous course of antibiotics, (c) the probable fact that most existing bacteria don't grow on conventional culture media, and (d) the probable fact that there is a significant number of uncharacterized bacteria that infect human tissue.

Krieger et al. reason that these problems could be overcome through molecular approaches. They applied an approach in which DNA is detected and characterized.
John Garst

Date: Tue, 17 Dec 1996 20:34:12 GMT

From: Jeff Parker

Subject: Re: What the Krieger article says

It appears that the study published by Krieger et. al.would imply that EPS culturing is a waist of time,based on the following:EPS "may acquire organims during passage through the urethra";the presence of inhibitory substances in EPS and previous antibiotic treatment may yield no growth;most bacteria do not multiply on conventional culture media;and bacteria commonly cultured and treated at present,e.g.S. Aureus and S. Epidermidis,appear to be "normal flora",present in none of the prostate tissue specimens examined in the study.In short,culturing EPS gives no reliable info. concerning what bacteria are actually present in the prostate,and may even muddy the water in terms of treatment selection.

It is interesting to note that the study was funded by the NIH;I understood from info. received from the Prostatitis Foundation that no such funds were available for prostatitis research.Am I missing something?

Date: Fri, 20 Dec 1996 14:11:27 -0500

From: John Garst

Jeff makes good points here, but I'm not sure that I agree entirely.

  1. In their screening, Krieger et al. used the usual "first shot" EPS, not that obtained after several prior drainages. Thus, their EPS samples were not necessarily adequate.
  2. I suspect that they regarding "normal skin flora" found in cultures as contaminants, as is the usual practice. If so, then even if they found Staphs in the EPS cultures, they ignored them.
  3. There are other studies that document prostatic Staph infections.
  4. The only specific mentions of Staphs that I find in the Krieger paper refer (a) to a group of 12 patients with skin samples positive for Staphs (which were *not* found in the prostate biopsies of *these 12 patients*) and (b) the appearance of Staphs on an "unrooted dendrogram obtained by using the Phylip phylogenetic inference program ["The separation of the group consisting of S. aureus, S. epidermidis, and 5744 (skin biopsy), from the grouping of patient prostate biopsy sample sequences, is highly significant (98% confidence)." This, of course, still refers to just those 12 patients.
  5. "A total of 29 clones from 10 patients were sequenced completely or partially" (in order to compare base sequences with known sequences for individual bacterials species). This is a relatively small number of patients.
  6. None of these 10 had prostatic Staph.
  7. One of the 10 had E. coli in the prostate (a normal gut bacterium).
  8. We don't know what the other 103 subjects who were positive for prostatic bacteria had. Staph has not been ruled out. One can argue that if it were commonly the pathogen, it should have been found in one or more of the 10.
  9. Five unidentified prostatic bacteria were found (labeled "15735," E2Com," "15751," "C5752," and "15725"). We know nothing of the culture/sensitivity behaviors of these bacteria. Since bacterial identification in cultures is based on specific tests of various sorts, we don't know that any of the tests commonly used would separate these from known species. My guess is that they might not. Thus, Feliciano and others culturing EPS and finding various species, such as Staphs, could really be finding these unknown bacteria and misidentifying them. Whatever, a sensitivity does not depend on identification. It gives the information you want (what antibiotic will work) even if the identification is wrong.
  10. "For each patient, at least two prostate biopsy specimens were evaluated by each molecular assay..." This isn't very many, and the contents of particular occluded acini could have been missed easily.
For these reasons, I don't think that the Krieger study "completely undercuts the EPS bacterial culturing basis of the [Feliciano] protocol." So the bacteria are misidentified maybe? So what?
John Garst

Date: Sun, 22 Dec 1996 01:57:16 GMT

From: Jeff Parker

Subject: Landmark Prostatitis Study--offers to cure

This post is in reply to John Garst's post responding to my initial post concerning whether the recent study published by Krieger et. al. supports an argument for the use of the so-called Feliciano protocol.John raised several important issues regarding the study,the primary ones relating to the small number of samples used for certain aspects of the study and answers to questions the study leaves open.

Perhaps John's concerns can be addressed by rephrasing my original post.I personally would not include the Krieger study in my Feliciano protocol sales brochure;the study's authors summarize several shortcomings of EPS culturing(e.g.EPS picking up bacteria from the the urethra and elsewhere and difficulties in culturing because of inhibitory substances,prior antibiotic use,and culture media problems)and no staph, an organism nearly always cultured by Feliciano himself,was discovered by the authors in any patients where bacterial DNA sequencing was completed nor in those patients whose skin biopsy specimens were compared to their prostate biopsy specimens.Quite frankly,knowing what I do now and learned the hard way, I wouldn't waste my time preparing such a brochure for a treatment no more successful than doing nothing.

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