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The authors studied the survival of Gardnerella vaginalis in human urine and determined conditions for optimum recovery on agar media. Gardnerella counts declined by greater than 99.9% in urine held at 37 degrees C for 24 hours, whereas the falloff was negligible at 4 degrees C. Viability was lost after 6 hours in urine with pH of 5, and only 0.01% cells survived in urine with pH of 7. In contrast, greater than 90% cells survived exposure at pH of 6. Dialysis to remove small molecular weight (less than 14,000) inhibitors did not enhance survival. Co-cultivation with Ureaplasma urealyticum and the addition of glycogen improved survival. Maximum recovery from urine required anaerobic incubation on enriched agar medium (pH 6.5-7.5) for at least 48 hours. Gardnerella vaginalis survives poorly in human urine at 37 degrees C. Culture for these bacteria requires prolonged anaerobic incubation.

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Ureaplasma urealyticum has previously been shown to be capable of persisting in the rat kidney for up to 6 months following a single reflux challenge. We examined kidney tissue from infected animals for evidence of renal damage by using standard cytochemical and immunoenzyme methods. We also monitored changes in renal function during a 6-month study period with standard biochemical assays of plasma and urine. Histologic examination showed tubular atrophy, interstitial fibrosis, and a mononuclear infiltrate in proportion to ureaplasma counts from renal tissue. The most severe damage was accompanied by hyaline cast formation within tubules which gave rise to the typical thyroidlike appearance of chronic pyelonephritis involving conventional urinary pathogens. Macroscopic renal scarring occurred in some animals. Although damage to the renal medulla was moderate to severe, only minor changes were seen in the cortex, and glomeruli were invariably spared. Biochemical tests of renal function showed similar changes in infected and uninfected animals during the study period. Interstitial inflammation was characterized by a mononuclear cell infiltrate in which polymorphonuclear leukocytes were not conspicuous. It is evident that U. urealyticum is capable of producing chronic pyelonephritis in the rat after a single reflux challenge. The results of this study have obvious implications for the pathogenicity of these bacteria in human pyelonephritis.

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Nucleated nonsquamous cells in urine of patients with crescentic glomerulonephritis (CN), noncrescentic glomerulonephritis (NCN), acute tubular necrosis (ATN) and drug related acute interstitial nephritis (AIN) were identified using monoclonal antibodies and immunoperoxidase stain. Cell viability was determined by trypan blue permeability. CN was distinguishable from NCN by total cell numbers exceeding 30,000/ml (p less than 0.001) and counts of granulocytes exceeding 10,000/ml (p less than 0.05), monocytes exceeding 3,000/ml (p less than 0.001), T4 lymphocytes exceeding 1,500/ml (p less than 0.001), T8 lymphocytes exceeding 1,500/ml (p less than 0.001), glomerular epithelial cells exceeding 4,000/ml (p less than 0.001), proximal tubular cells exceeding 8,000/ml (p less than 0.001), loop of Henle cells exceeding 1,500/ml (p less than 0.01) and urothelial cells exceeding 1,500/ml (p less than 0.05). AIN was distinguishable from ATN by total cell numbers exceeding 75,000/ml (p less than 0.001) and counts of granulocytes exceeding 150,000/ml (p less than 0.001), monocytes exceeding 5000/ml (p less than 0.001), T4 lymphocytes exceeding 3,000/ml (p less than 0.01), T8 lymphocytes exceeding 2,500/ml (p less than 0.01) and cell viability exceeding 60% (p less than 0.05). Proximal tubular, loop of Henle, distal tubular/collecting duct and urothelial cells were present in high numbers in CN, ATN and AIN. CN can be distinguished from NCN, and ATN can be distinguished from AIN by identifying and quantifying the nucleated cells present in the urine.

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A challenge procedure based on reflux of Ureaplasma urealyticum to the rat kidney was used to examine the disease-causing potential of this species for the upper urinary tract. Renal infection was detected 8 days after challenge in 90 to 100% of animals exposed to 10(7) ureaplasmas but was not detected in animals exposed to fewer than 10(6) microorganisms. Viable microorganisms were recoverable from more than 60% of infected animals throughout a 24-week follow-up period of study. Animals with persistent infection exhibited a humoral response involving immunoglobulin M and immunoglobulin G antibody. These results show that U. urealyticum is capable of giving rise to chronic infection in the kidneys of rats following challenge via the ascending route.

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Controversy exists as to the type of cells present in the urine during renal allograft rejection. In order to resolve this controversy as well as to evaluate the value of urine sediment examination as a means of detecting AR, we quantitated the different cells present in urine during AR using an immunoperoxidase technique and monoclonal antibodies reactive with lymphocytes, monocytes, granulocytes, glomerular epithelial, tubular, and urothelial cells. Urine sediment (n = 176) was examined serially over 3 months in 15 transplant recipients. There were 12 episodes of early posttransplant acute tubular necrosis and 21 episodes of AR. It was possible to detect AR as well as to distinguish AR from ATN. Lymphocyte and tubular cell excretions were increased significantly during AR. Excretion of urothelial cells was also significantly increased during most episodes of AR suggesting that rejection of ureters occurs concomitantly with rejection of the kidneys.

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Quantitative culture of midstream urine fails to yield a significant microorganism in many patients with acute urinary symptoms. We cultured bladder urine, obtained by aspiration, from symptomatic adults with equivocal findings on standard testing of midstream urine for low numbers of conventional uropathogens and fastidious bacteria. We found 561 (31%) of 1817 women and 36 (12%) of 300 men to be culture positive. Five hundred eighty-one (70%) of 830 isolates were fastidious bacteria; 191 (34%) of 561 culture positive women and 0 of 36 culture-positive men had polymicrobic bacteriuria. Bacterial counts were less than 10(5) colony-forming units/mL in 67% of samples; 204 of 406 patients with single-species infections had increased leukocyte counts in urine. Patients with symptoms of urinary tract infection who are culture negative on standard testing may harbor fastidious bacteria or low numbers of conventional uropathogens in the bladder. In these patients, culture of bladder aspiration urine for low counts and fastidious species is necessary to diagnose bacteriuria.

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A method to identify nucleated nonsquamous cells in urine using monoclonal antibodies and immunoperoxidase stain is described. Cells from washed deposits of midstream urine samples were transferred to gelatinized slides in a cytocentrifuge, air-dried, acetone fixed, and subjected to microwave irradiation. Slide preparations were then treated with monoclonal antibodies with the use of a four-layer peroxidase-antiperoxidase technique. It was possible to identify granulocytes, monocytes, lymphocytes, and renal epithelial and urothelial cells. This method was found to be helpful in determining the profiles of cells in urine in acute tubular necrosis, drug-related acute interstitial nephritis, and crescentic glomerulonephritis.

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Accurate identification of nucleated cells in urine can be difficult with conventional methods of microscopic urinalysis. Monoclonal antibodies were used with an immunoperoxidase technique to identify nucleated cells in urine. This new development in urinalysis is in its early stages, but it has helped to circumvent the difficulties associated with standard microscopy. The monoclonal antibody technique allowed for the identification of granulocytes, monocytes, lymphocytes, glomerular epithelial, proximal tubular, loop of Henle, distal tubule/collecting duct, and urothelial cells in urine, and by quantifying these cells it was possible to determine the urine cell profiles in various renal diseases as well as in allograft rejection and early post-transplant acute tubular necrosis in renal allograft recipients. The cell profiles are useful in aiding the diagnosis of these conditions.

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Midstream urine samples from 106 patients presenting to the Casualty Department of The Royal Melbourne Hospital with frequency or dysuria were cultured for Gardnerella vaginalis and conventional uropathogens. Urine samples collected via an open-end catheter from 70 healthy pregnant women were examined similarly. Midstream urine and other samples, including the seminal fluids and swabs of the mouths, throats, rectums, and vaginas of 33 healthy subjects, were cultured for G. vaginalis. Another 15 female patients with proven G. vaginalis bacteriuria were given a bladder washout localization test to determine the site of infection in the urinary tract. G. vaginalis in counts greater than 10(3) CFU/ml was recovered from the midstream urine of 27 of 106 patients (25%), 7 of whom also harbored conventional pathogens in counts greater than 10(4) CFU/ml. Another 11 patients with cultures negative for G. vaginalis yielded greater than 10(4) CFU of conventional pathogens per ml. G. vaginalis was cultured (greater than 10(3) CFU/ml) from catheter samples of 19 of 70 healthy pregnant women (27%), 6 of whom also harbored greater than 10(3) CFU of conventional uropathogens per ml. Two women yielded growths of conventional pathogens only. Midstream urine samples from 13 of 13 healthy males were free of G. vaginalis, whereas 5 of 20 healthy nonpregnant females yielded greater than 10(3) CFU of G. vaginalis per ml from midstream urine samples. G. vaginalis was recovered from 4 of 12 semen samples and from urethral samples from four of seven males and four of eight females. All four culture-positive females also harbored G. vaginalis in their vaginas. There was no evidence of oral or rectal carriage of G. vaginalis in 15 healthy subjects. Localization studies with 15 female patients having underlying renal disease showed that 11 patients harbored G. vaginalis in their kidneys. The result suggest that colonization or infection of the bladder and upper urinary tract by G. vaginalis is very largely a phenomenon of females, with the highest frequency in pregnant women. The prevalence of G. vaginalis in the urinary tracts of healthy females is similar to that of symptomatic subjects. However, G. vaginalis in counts greater than 10(5) CFU/ml is more likely to be associated with urinary tract symptoms. In males, this bacterial species infects the genital tract rather than the urinary tract.

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We examined the occurrence of crystals and casts in the urine of healthy subjects after administration of triamterene and the site of crystal formation in experimental animals. Twenty out of twenty healthy subjects had abundant triamterene crystals and casts in acid urine after receiving a single 100 mg dose. Casts were present in the urine from 2-11 hours after administration of the diuretic. Cast formation occurred in acidic urine and was prevented by alkalinization of the urine with potassium citrate. Animal studies showed that crystallization and cast formation occurred in the medullary and papillary collecting ducts of the rat kidney. These findings provide a possible explanation for the reported nephrotoxicity of triamterene, particularly when given to patients who are receiving non-steroidal anti-inflammatory agents.

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Ureaplasma urealyticum cultures from 124 patients with urinary tract disease were serotyped by indirect immunofluorescence, using antisera to serotypes I to VIII. A similar range of serotypes was recovered from first-voided, midstream, and bladder-aspiration (SPA) urine, upper urinary tract samples, and vaginal swabs. Serotype VI was predominant (44/124) among the samples, whereas serotypes V (1/124 samples) and VII (0/124 samples) were uncommon. Twenty of 124 cultures contained more than one serotype, and three cultures were untypeable. Serotypes cultured from bladder urine were also present in vaginal and urethral samples, although these samples often carried additional serotypes. Consecutive SPA samples from the same patient invariably contained the same serotype, whereas some consecutive midstream urine samples showed a loss or gain of serotypes with time. One patient carried the same serotype in SPA urine over a period of 13 months. The pattern of serotypes recovered from the urinary tract was similar irrespective of the sampling site, the site of infection, the clinical diagnosis and renal function of the patient, and the presence or absence of other microorganisms. Colonization above the urethra and association with urinary tract disease appeared to be serotype independent.

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Colony counts were compared from urine samples obtained by suprapubic aspiration and via a short, wide bore, open-ended urethral catheter. We studied 30 female patients in whom suprapubic aspiration of bladder urine was necessary to confirm the presence of bacteriuria with conventional or fastidious organisms. Catheterization was done immediately following suprapubic aspiration. Culture results of mid catheter urine specimens were similar to those from suprapubic aspiration urine specimens in 27 of 30 patients, a result considerably superior to that obtained with a conventional side-hole catheter. We conclude that a short, wide bore, open-ended catheter should be used to obtain urine specimens from female patients. Results confirm catheter specimens to be a satisfactory alternative to bladder aspiration of urine for detection of bacteriuria caused by fastidious micro-organisms or in patients with low numbers of conventional urinary tract pathogens.

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Urinary casts from 46 healthy volunteers and 60 patients with glomerulonephritis were examined for the presence of Tamm-Horsfall glycoprotein and other proteins. All samples gave immunofluorescence evidence of Tamm-Horsfall protein in casts. Casts from 59 of the patients but only three of the controls contained other proteins in addition (p less than 0.001). Immunoglobulins (IgG, IgM, IgA) were detected in casts from 53 of the patients but none of the healthy volunteers. Examination of urinary casts for immunoglobulins, complement, and fibrin provides a non-invasive method for distinguishing patients with active glomerular disease.

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Urine samples from 141 consecutive patients referred for investigation of microscopic hematuria were examined by phase-contrast microscopy to determine the probable site of bleeding into the urinary tract. Dysmorphic (i.e. morphologically variable) erythrocytes, suggestive of glomerular bleeding, were present in 86 of 87 patients with significant hematuria who were later shown to have glomerulonephritis. In contrast, all of 30 patients with non-glomerular lesions had isomorphic (i.e. morphologically uniform) erythrocytes in the urine. Ten patients yielded a mixed morphologic pattern suggestive of dual pathology which was confirmed in four patients. Urine erythrocyte counts from 376 healthy individuals showed that 95% yielded less than 8,000 cells/ml and in each case erythrocytes exhibited a dysmorphic pattern suggesting that erythrocytes enter the urine of healthy subjects via the glomerulus. Electron microscopy of urinary erythrocytes from 36 patients with glomerulonephritis demonstrated the range of dysmorphic changes characteristic of glomerular bleeding, presumably resulting from environmental changes suffered by the erythrocytes within renal tubules.

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Bladder aspirate urine samples (N = 190) were cultured for the presence of fastidious microorganisms. These samples were obtained from patients with urinary tract disease in whom standard bacteriologic investigation had failed to indicate infection. Gardnerella vaginalis was recovered alone, or in association with Ureaplasma urealyticum from the bladder urine of 33% of patients with reflux scarring. G. vaginalis was localized to the upper urinary tract in 75% of these patients with bladder counts greater than 10(3) colony-forming units per milliliter. The results show that two fastidious microbial species, not conventionally associated with urinary tract infection, are recoverable from the upper urinary tracts of patients with so-called sterile pyelonephritis.

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