The direct microscopy and the culture methods each have pitfalls In the past 20-25 years the direct visualization of bacteria in urine has largely been abandoned in favor of the methods involving culturing and counting the colonies of bacteria. Indeed virtually all of the studies of the significance of bacteriuria are based upon culturing the urine, and the direct microscopic examination of urine has been relegated to the status of a quick but inadequate screening procedure which may be helpful because it can be correlated with the culture methods.
Any culture method requires that the bacteria will grow in the laboratory in the medium selected and in the time allotted. If the bacteria are damaged or dead when they left the body, then they will not grow There are many reasons why bacteria in urine would be damaged. The ionic strength or osmolality of the solution may be damaging. The oxidation potential of urine is usually too high (e.g., +0.22 to +0.25 volts). There may be a noxious metabolite in urine. (Human antibodies have been identified in urine and they have been demonstrated to be deposited on bacteria in urine.) Any or many of these factors may render a given bacterium non-viable in vitro. Finally, if the medium used is inappropriate for the growth of the particular organisms present, they will not grow. It can be readily shown by staining and microscopy that many of the bacterial forms found under the microscope were not alive at the time the specimen was obtained. For example, some do not contain nucleic acid, a biochemical component essential to life. Should all of the bacteria in a given specimen be devoid of nucleic acid, then none will grow and the culture of urine remains sterile. Indeed many urine specimens from sick patients containing huge numbers of bacteria will not yield a thriving bacterial culture in the hospital bacteriology laboratory. When the laboratory reports "no growth" the clinician may abandon the possibility of significant bacteriuria, and hence the possibility of an infectious cause. Nonetheless, these dead, damaged or fastidious bacteria, though they do not grow in culture, may in vivo have caused or exacerbated the patient's illness. FIG. 2 shows innumerable small cocci found, using the novel method taught herein, in the urine of a patient for whom the attempt to demonstrate bacteriuria by culture technique was completely unsuccessful. With regard to the direct examination of the urine, it must be noted that although bacteria may be seen in urine at only 100 diameters magnification, the size of the image is not the only consideration. Should the optical density and refractive index of a dead bacterium be near that of the medium, then it would not be detected by ordinary light microscopy. It may be seen by staining or perhaps by some specialized lighting. (Even then, as pointed out by Kunin, round bacteria cannot be distinguished from other near round particles such as crystals.) In my method the urine is examined wet at 100 to 400 diameters magnification, but it is also dried and prepared in a particular way so as to retain and preserve the bacterial structure through staining. In particular, I have found that urine contains lipids which act as detergents. Should they be allowed to remain on the slide when an aqueous dye is applied to the slide, then much of the sediment (including bacteria) will wash off of the slide and the preparation will be lost. This is a major reason why past attempts to study bacteriuria have failed. The photograph of FIG. 1 illustrates the difference. In that photograph one slide had been prepared in the standard way and the other has been washed with a lipid solvent. After staining much more sediment is found on the washed slide. Most of the sediment had washed off in the standard preparation. A chromatogram of the lipids removed reveals several lipids in the range of polarity of the phospholipids (e.g., lecithin, phosphatidylserine, etc. which substances act as detergents) but they do not contain appreciable phosphorus and thus they are not phospholipids. Standard methods of preparing and staining urine specimens, such as that of Melnick, U.S. Pat. No. 4,225,669, do not provide for the precautionary removal of these lipids.
Rheumatoid arthritis (RA) is a chronic inflammation of the joints, generally regarded as a systemic autoimmune disorder. Its etiology is unknown, but it has been postulated that it is associated with microbial infection. See, e.g., D. C. Demonde, ed., Infection and Immunity in the Rheumatic Diseases, 95-287 (Blackwell Scientific Publications, London: 1976). The evidence, however, until the present discovery, was inconclusive. See, e.g., D. J. McCarty, et al., ed., Arthritis and Allied Conditions: A Textbook of Rheumatology, ch 28 at 417 (9th ed. 1979); R. G. Petersdorf, et al., ed., Harrison's Principles of Internal Medicine, Part Six, Chapter 346, at 1977 (McGraw Hill: 1983). Bacteriuria has not been associated with RA, and indeed one authority remarks "Urinary abnormalities are relatively uncommon in RA . . . Urinary tract infection was not found to be increased in RA patients." McCarty, supra, chapter 33, page 499, citing Ann. Rheum. Dis., 27: 345 (1968). Hypertension is a chronic elevation of blood pressure resulting from the obstruction of blood flow within the kidney (secondary hypertension) or without apparent cause (essential hypertension). One kidney disorder associated with secondary hypertension is pyelonephritis, the inflammation of the renal pelvis of the kidney as a result of bacterial infection, usually responsive to antibiotics. It has not been reported, however, that there is any correlation between essential hypertension and asymptomatic bacteriuria (bacteriuria observed in patients not reporting symptoms of urinary tract disorders). According to N. M. Kaplan, Clinical Hypertension, 14 (3d. ed. 1982), bacteriuria is found in 2-5% of hypertensives. Most of these positive cultures were of gram-negative rods. The method of the present invention has demonstrated a much higher incidence of bacteriuria in hypertensives, perhaps as high as 90%, and that cocci or "exploded cocci" are found in considerable numbers.