Cystitis Research Center

For sake of these victims, the medical professional and its "detective" agency can and must return to the valid concepts of classic testing technology. According to the Health Care Financial Administrator Rules, the laboratory must honor the physicianÕs request for PURE CULTURE diagnostic technology from the laboratory, and the laboratory must diligently screen all specimens for evaluation of ALL organisms present to find the true causative agent, including the above Gram-Positive pathogens. We have knowledge and capability and are obligated as responsible medical scientists to use the most effective technology available.

The Cystitis Research Center welcomes your comments in the technical issues of diagnostic technology.

Sincerely yours,
Paul Fugazzotto, PhD.

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The CRC Answer To The Colony Count

The laboratory studies at the Cystitis Research Center (CRC) for evaluation of pelvic disorders, began in 1964 with a focus on devising a test procedure analogous in principle to those used universally in diagnosis of all disease entities (viral, bacterial, fungal parasitic).

The identity of each condition is determined by manipulations specific for isolation and identification of the respective etiologic agent. At the CRC, we consider the medical laboratory as the Detective Agency for the medical profession, analogous to the Police Detective in the social area of problems.

A good analogy in point is at the airport facility where the uniformed agents do not merely count the number of people passing to the boarding area (on basis of appearance) to estimate possible antisocial "characters"; they are obligated to scrutinize each individual very closely to detect evidence of a "social pathogen": the "character" with a gun, long blade, bomb, or other weapon.

We of the microbiology lab are also obligated, with valid technology to scrutinize the test sample for the respective PATHOGEN, among the many organisms passing our view. This is not accomplished by a casual superficial colony count, especially one focused merely on fecal bacilli, disregarding the well recognized pathogens present, established as such for over 100 years. Here, at CRC we have the background of 58+ years, scrutinizing all manner of specimens for specific pathogens. The etiologic agents of pelvic disorder respond to the same principles as do all those of other disease entities. This is the focus of the CRC program, which now has the support of studies involving more than 22,000 clinical specimens, tested in duplicate for comparison of PLATE COUNT vs. PURE CULTURE technology. Certainly sufficient data to validate the CRC findings: strains of Gram-positive cocci as etiologic pathogens.

Doctors and patients alike must insist that the "COLONY COUNT" practice be immediately discontinued, and that medical labs learn and adopt the only valid approach to evaluation of pelvic infection: PURE CULTURE TECHNOLOGY (which requires initial culture of specimens in broth, to allow all microbial strains from the specimens to emerge,) Then by transfer of aliquots to appropriate differential media, the etiologic agent can be isolated for further study, and be reported to the attending physician along with the appropriate antibiotic sensitivity pattern. Plus, the physicians' prescriptions no longer becomes a hit and miss proposition.

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Laboratory Test Technology For Diagnosis And Monitoring Of Urinary Tract Disorder

Update and revision of the "Method for Revealing Hidden Pathogens"; in Laboratory Management: 24-29, Oct. 1987; A clinical laboratory diagnostic procedure.

Test #1: Test for Presence of Antibiotic in the Urine

1. This is done on all specimens, initial to the program, in order to detect evidence of false reactions due to extraneous components antagonistic to recovery of offending organisms.
2. This is also done periodically on participant urines, to monitor the antibiotic treatment program on previously diagnosed study participants.
3. Utilizing a Trypticase Soy Agar (TSA) Plate, with a crayon line drawn on the plate bottom, to divide the plate into halves: Apply a suspension of E.coli (Gram-negative) to one-half of the agar and a suspension of Enterococci (Gram-positive) to the other half. Spread each out for confluent growth.
4. Utilizing two _" blank disks immersed in each urine specimen, place one disk onto the E.coli side and the other disk onto the enterococci side of the plate. Incubate plate at 35¡C.
5. Determine if antibiotics are present by observing inhibitory zones around the discs. Measure the zones in mm and document. As many as 6 specimens can be tested per plate.
6. In a chart such as the following, record the size of the inhibitory zone in mm observed for each specimen on its tests, indicating the amount of antibiotic excreted into the bladder.

Evaluation of Results

The value recorded in the G+ test is used in the evaluation of patient response.

The result in the G- is used as a negative control.

Wet Prep Procedure (optional)

This test is done in anticipation of viewing the organisms to be expected as emerging from transfer of the broth culture; or to confirm the result of the broth transfer.

Transfer one loop of each broth to a microscope slide. Cover with cover slip and examine at 430X magnification to record the nature and morphology of the organisms present, i.e., strep, other cocci, motile and non-motile bacilli, other strains. Although the sub-culturing can be made to the respective differential media, prior to examinations of wet mounts, cocci in the presence of other organisms may be missed if one is not aware of their presence in the broth, and fails to make special efforts to isolate and identify them.

Diagnostic Procedure

I. Purging of the raw specimen.

1. All raw urines voided by the patients can be expected to contain body waste products from foods, ingestibles, medications (antibiotics), toxins; antagonistic to the emergence of offending pathogens. Aliquots of all urines must first be purged of these aqueous soluble components.
2. Using a cotton tipped applicator, stir the urine well, and with the saturated swab transfer one or two portions of the urine to a 13 x 100 ml screw cap tube containing 5 or 6 ml of sterile saline solution. The saline solution should consist of 0.85 g NaC1, plus 0.1 gram proteose-peptone as a buffer per 100 ml distilled water. Securely replace the cap. Hold tube in an upright position, and pass it with smooth inversion excursions by twist of the wrist, completely inverting two times per second, for 10 seconds (20 complete inversions).
3. Centrifuge the tube at 2,000 rpm for 20 minutes. Pour the supernate off; add 3.0 ml of Trypticase Soy Broth (TSB) to the residue and replace the tube cap. Shake the tube briefly and incubate at 35¡C until growth appears (usually in 3 days), or for as long as 6 days if negative in 3 days. It is convenient to have a supply of TSB in 13 x 100 mm screw cap tubes already prepared for the transfer.

II. Isolation of the Offending Organisms

1. To isolate the offending organisms, it is necessary to employ agar plates of differential media: Phenyl Ethanol blood agar (PEA) and Eosin Methylene Blue (EMB) agar. The plate bottoms are marked with wax crayon, in as many as 8 segments with lines crossing at the plate center, depending on the numbers of cultures to be processed.
2. With a 0.1mm wire loop, transfer an aliquot of each culture to respective segments of both plates; but carefully spread it on the PEA for distinct colony isolation.
3. After inoculation of the PEA segments, puncture the agar for possible hemolysis. Place a NA disc (Negram) on the PEA surface, where the marks cross at the center. Incubate both plates at 35¡C for about 24 hours or until significant bacteria emerge on the plate.
4. Determine which prominent discrete organisms are significant on the PEA agar plate in the area close to the NA disk. Find one or more totally isolated, completely round colony on the plate, making sure that it has no imperfections such as mixed colony growth.
5. Utilizing a straight wire, transfer and spread the chosen colony from the PEA plate to a separate routine blood agar plate. Puncture this blood plate a few times to show its hemolytic characteristic. Incubate at 35¡C overnight and observe growth to ensure a pure culture confluent growth (with no breakthrough of submerged colonies).
6. The EMB (Gram-negative) plate culture growth is identified merely for the record, as these organisms are not considered significant of infection. (UTI/IC are Gram-positive cocci infections.) The Gram-negative produces discomfort by fermentation.

III. ABST (Antibiotic Sensitivity Test) of infecting Gram-positive pathogens.

1. After the infecting organism is identified, perform its ABST (Antibiotic Sensitivity Test) on the pure culture. Utilize only the agar diffusion technique. (Do not use the multiple plastic well plate method.)
2. From the confluent growth in step 5 above, prepare a light saline suspension of the etiologic agent with a sterile cotton tip applicator.
3. Transfer this suspension sparingly over the surface of a TSA plate, streaking at two right angles for confluent growth.
4. Space the antibiotic discs equally on the agar surface.
5. Invert the plate and incubate at 35¡C for overnight (16-20 hrs.).
6. Read and record the diameters of the inhibitory zones around each antibiotic.

Antibiotics Used, and Their Respective Manufacturers Minimal Inhibitor Concentration For Gram-Positive Pathogens

1. In our reporting, for purpose of patient treatment, we do not consider an antibiotic effective for treatment, unless the antibiotic reading in the test, amounts to 5mm and higher than the manufacturer MIC value.
2. The organisms emerging on the EMB are not pathogenic, but do cause some discomfort by reason of fermentation. They also require treatment.
   1. Gram-negative (intestinal) bacilli respond to customary Cipro (and other similar antibiotics).
   2. Yeasts respond to Nystatin, Diflucan, and other anti-yeast treatment.

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