- Email: [email protected]
STREPTOCOCCI AS URINARY PATHOGENS
479
vascular resistance are not mutually exclusive forms of renal impairment. The clinically observed reversible renal impairment may be more closely related to the latter, whereas previously reported irreversible renal damage in cyclosporin-treated patients21,22 may be evidence of longterm effects of tubular toxicity. The methods by which cyclosporin causes renal vasoconstriction are unknown. Nonetheless, Siegl et al14 believe that the vascular effect (as opposed to renal tubular effects) is the prime mechanism of the renal impairment. Animal studies have suggested a role for a cyclosporinstimulated renin-angiotensin system, 14,23 whereas human studies19.24 have suggested that the renin-angiotensin system is actually suppressed by cyclosporin. Our studies did not address this question, but mean arterial pressure was significantly higher in our prospective study patients before conversion from cyclosporin to azathioprine. Non-reninmediated post-transplantation hypertension is rare in our experience, and we believe the role of the renin-angiotensin system in cyclosporin-induced post-transplantation hypertension needs further study. It is apparent that the cyclosporin-induced rise in vascular resistance is reversible, when patients are converted to azathioprine after nearly a year of treatment. It is not certain, however, whether longer exposure to cyclosporin might lead to permanent damage to the allograft. Prolonged renal vasoconstriction could cause tubular damage and eventual fibrosis and chronic nephron loss. This effect would be difficult to establish in kidney transplant patients, since chronic rejection could also cause chronic nephron loss. Even among large cardiac transplant centres the evidence that cyclosporin causes chronic irreversible renal damage is debated.25 A better understanding of the way cyclosporin causes renal vasoconstriction might reveal other, perhaps pharmocological, manipulations that could reverse its haemodynamic effect. It might also be possible to use a reduced dose regimen of cyclosporin that maintained the immunosuppressive effects but did not raise renal vacular resistance. If the renal vasoconstrictive effects of cyclosporin are, indeed, dose related the trend for some centres to use triple therapy with prednisone, low-dose cyclosporin, and low-dose azathioprine regimens may gain further support. This study was supported by a grant from the General Clinical Research Center (RR-0032) National Institutes of Health.
Corespondence should be addressed to J. J. C., Univesity of Alabama at Birmingham, Nephrology Research and Training Center, Birmingham, Alabama 35294, USA. REFERENCES 1. The Canadian Multicentre Transplant Study Group. A randomized clinical trial of cyclosporine in cadaveric renal transplantation. N EnglJ Med 1983; 309: 809-12. 2. European Multicentre Trial Group. Cyclosporin in cadaveric renal transplantation: one year follow-up of a multicentre trial. Lancet 1983; i: 986-90. 3. Bennett WM, Pulliam JP. Cyclosporine nephrotoxicity. Ann Intern Med 1983; 305: 267-73. 4. Chapman JR, Griffiths D, Harding NG, Morris PJ. Reversibility of cyclosporin nephrotoxicity after three months’ treatment. Lancet 1985; i: 128-30. 5. Flechner SM. Cyclosporin therapy. Transplant Immunol 1984; 1: 1-4. 6. Tauxe WN, Dubovsky EV, Kidd T. New formulae for the calculation of effective renal plasma flow. Eur J Nucl Med 1982; 7: 51-54. 7. Dubovsky EV, Russell CD. Quantitation of renal function with glomerular and tubular agents Semin Nucl Med 1982; 12: 308-29. 8. Lustig S, Silis V, Berger ME, et al. Effect of low dose cyclosporin A on blood pressure and arterial contractility in spontaneously hypertensive rats (abstract). American Society of Nephrology, New Orleans. December 15-18, 1985: 102A. 9. Moss NG, Powell SL, Falk RJ. Intravenous cyclosporine activates afferent and efferent renal nerves and causes sodium retention in innervated kidneys in rats. Proc Natl Acad Sci USA 1985; 82: 8222-26. 10. Gazdar AF, Dammin GJ. Neural degeneration and regeneration in human renal transplants. N Engl JMed 1979; 283: 222-24.
STREPTOCOCCI AS URINARY PATHOGENS LYNN E. COLLINS ROSEMARY W. CLARKE ROSALIND MASKELL Public Health Laboratory, St Mary’s Hospital, Portsmouth PO3 6AQ
In a 2-month prospective study of streptococci isolated from urine specimens in the laboratory, 242 strains of catalase-negative gram-positive cocci or coccobacilli were isolated in substantial numbers from 11 725 specimens. These comprised 10% of the important isolates. Species identification of all isolates was undertaken. 74 (30%) of the isolates were of species other than Streptococcus faecalis and S agalactiae. 79 (33%) were not detected on cysteine-lactose-electrolyte-deficient agar after overnight incubation in a carbon dioxide incubator. 20 of the 24 isolates of coccobacilli were Gardnerella vaginalis. Many of the isolates of fastidious species were accompanied by pyuria. An isolation protocol practicable in busy laboratories is proposed.
Summary
Introduction OVERNIGHT aerobic culture of urine specimens on primary isolation media leaves the symptoms of many patients and much pyuria unexplained. There has been interest in the pathogenicity of the fastidious species of streptococcil and there have been occasional reports of their isolation from midstream urine samples of patients with urinary-tract infection2 and from bladder urine of catheterised patients.3 We have undertaken a prospective study of streptococcal urinary infection, using methods capable of detecting such organisms. Some fastidious streptococci can appear coryneform (coccobacillary) in gram-stained preparations made from cultures on solid media;4 so that such species should not be missed we included all catalasenegative gram-positive cocci and coccobacilli in the study. Dieperink H, Starklint H, Leyssac PP. Nephrotoxicity of cyclosporine—an animal model: study of the nephrotoxic effect of cyclosporine on overall renal tubular function in conscious rats. Transplant Proc 1983; 15 (suppl 1): 2736-41. 12. Humes HD, Jackson NM, O’Connor RP, Hunt DA, White MD. Pathogenetic mechanisms of nephrotoxicity: insights into cyclosporine nephrotoxicity. Transplant Proc 1985; 17: 51-62. 13. Finn WF, Gitelman HG. N-acetyl-B-glucosaminidase excretion during cyclosporin A 11.
Human Toxicol 1984; 3: 416. et al. Cyclosporine, the renin-angiotensin-aldosterone system, and renal adverse reactions. Transplant Proc 1983; 15 (suppl 1): 2719-25. 15. McKenzie N, Devineni R, Vezina W, Keown P, Stiller C. The effect of cyclosporine on organ blood flow. Transplant Proc 1985; 17: 1973-75. 16. Paller MS, Murray BM, Ferris TF. Decreased renal blood flow after cyclosporine infusion (abstract). American Society of Nephrology, Washington, DC. Dec 9-12, 1984: 248A. 17. Jao S, Waltzer W, Arveit LA. Acute cyclosporin induced decrease in GFR is mediated by changes in renal blood flow and renal vascular resistance (abstract). American Society of Nephrology, New Orleans, Dec 1985: 282A. 18. Sullivan BA, Hak LJ, Finn WF. Cyclosporine nephtrotoxicity: studies in laboratory animals. Transplant Proc 1985, 17: 1145-54. 19. Whiting PH, Simpson JG, Davidson RJ, Thomson AW. Pathological changes in rats receiving cyclosporin A at immunotherapeutic dosage for 7 weeks. Br J Exp Pathol 1983; 64: 437-44. 20. Gerkins JF, Bhagwandeen SB, Dosen PJ, Smith AJ. The effect of salt intake on cyclosporine-induced impairment of renal function in rats. Transplantation 1983; 38: 412-17. 21. Myers BD, Ross J, Newton L, Luetscher J, Perlroth M. Cyclosporine associated chronic nephropathy. N Engl J Med 1984; 311: 699-705. 22. Rao VK, Crosson JT, Kjellstrand CM. Chronic irreversible nephrotoxicity from treatment.
14.
Siegl H, Ryffel B, Petric R,
23.
cyclosporin A. Nephron 1986; 41: 75-77 Siegl H, Ryffel B. Effect of cydosporin on renin-angiotensin-aldosterone system. Lancet 1982; ii: 1274.
Bantle JP, Nath KA, Sutherland DER, Najarian JS, Ferris TF. Effects of cyclosporine on the renin-angiotensin-aldosterone system and potassium excretion in renal transplant recipients. Arch Intern Med 1985; 145: 505-08. 25. Porter G Discussion IV, diagnosis, incidence, and severity of cyclosporine nephrotoxicity. Transplant Proc 1985; 17: 232-44. 24.
480 TABLE II—CULTURE MEDIUM AND LENGTH OF INCUBATION
Methods Between Jan 1 and Feb 28,1986, we identified all the streptococci isolated from urine cultures on which sensitivity tests were carried out. The decision as to the importance of each isolate, and therefore whether to carry out sensitivity tests, was made in the same way as for all other urinary isolates. Factors taken into consideration included bacterial count, purity of growth, presence of pyuria (defmed as 10 cells/1 uncentrifuged urine), and relevant clinical details. Uncentrifuged urine was examined by the inverted microscope method. All specimens were inoculated onto cysteine-lactoseelectrolyte-deficient (CLED) agar by means of a 0-005 ml standard loop. Cultures were incubated overnight at 37°C in an atmosphere containing 7°o carbon dioxide. Any cultures that left either symptoms or pyuria unexplained after overnight incubation were reincubated for a further 24 h. Additional culture procedures were carried out on repeat specimens for which culture for fastidious organisms had been requested (usually because of a finding of apparently sterile pyuria on the previous specimen) and on specimens from patients with symptoms of prostatitis or epididymo-orchitis, in which fastidious organisms have been implicated.5 These specimens were also inoculated onto chocolated blood agar (CBA) and blood agar containing 16 J.lg/ml nalidixic acid (BAN). The CBA plate was incubated in 7% carbon dioxide and read at 18 and 48 h. The BAN plate was incubated for 48 h in an anaerobic cabinet (Don Whitley Mk II). All isolates of gram-positive, catalase-negative cocci or coccobacilli were identified by standard laboratory procedures-mannite fermentation on electrolyte-deficient agar (Streptococcus faecalis), orange coloration on Islam’s medium (S agalactiae), a strong caramel smell associated with sensitivity to penicillin (S milleri), growth anaerobically only with sensitivity to metronidazole (anaerobic streptococcus). Any organism not identified by these procedures was identified by means of API 20 STREP.
TABLE III-0RGANISMS ISOLATED FROM
30 SPECIMENS SHOWING
APPARENTLY STERILE PYURIA AFTER’OVERNIGHT INCUBATION ON CLED AGAR
specimens. 55% of the specimens showed pyuria, with the highest incidence (76%) in women of over 55 years. 24 isolates were coryneform on gram-stained film: 20 of these, including 3 from male patients, were Gardnerella vaginalis ; 3 S morbillorum; and 1 was a coryneform organism. Table II shows the numbers of isolates detected on CLED overnight incubation or after 48 h incubation or detected only on CBA and/or BAN plates. 30 specimens showed apparently sterile pyuria after overnight incubation on CLED agar. Table III shows the organisms subsequently isolated and the age and sex distributions of the patients from whom the specimens came.
agar after
Results
During the study period 11 725 urine specimens were examined. 2486 (21 %) isolates from these specimens were considered to be important, and of these 242 (10%) were included in the study. Table I shows the age and sex distributions of the patients from whom they were isolated. There were 71 isolates from 65 male patients and 171 isolates from 166 female patients. 81 (35%) of the patients were in hospital. 1 isolate was from a suprapubic aspirate, 17 from catheter specimens, and the remainder from midstream
Discussion
Although the duration of this study was short, the numbers of specimens examined and organisms isolated were large and some conclusions can reasonably be drawn. S faecalis and S agalactiae (Lancefield group B) are well-recognised urinary pathogens. 6 (5%) isolates of S faecalis and 11 (20%) of S agalactiae would not have been
TABLE I-ISOLATES OF CATALASE-NEGATIVE GRAM-POSITIVE COCCI AND COCCOBACILLI FROM URINE
Figures in parentheses number of specimens with pyuria. unidentifiable by the techniques used. =
*Isolated from
a
suprapubic aspirate.
tIsolated is mixed culture with S millen. tIsolates which were
481
detected after overnight incubation of cultures on CLED agar. 1 isolate of each organism grew only on additional media. The detection rates of the more fastidious species after overnight incubation of CLED agar cultures were much lower; 17 (74%) of isolates of S milleri, 25 (81%) of other streptococci, and 20 (100%) of G vaginalis required longer incubation. 43 (58%) of the 74 isolates of these species grew only on the additional culture media. It is not possible to say whether the detection rate would have been lower still if overnight incubation in air had been used, as it is for urine culture in most laboratories. However, it is very likely, at least, that the 12 isolates of S milleri and other fastidious streptococci that were detected after overnight incubation would have been missed. 3 isolates of streptococci, all S morbillorum, would have been missed if catalase-negative coccobacilli had not been included. It is interesting that both S agalactiae and G vaginalis were isolated from male patients; both are known to be 6 present in the female genital tract. Parker and Stringer pointed out that systemic infections with S agalactiae affect males as much as females, and Abercrombie and colleagues7 reported the isolation of G vaginalis from the bladder urine of a young man with acute cystitis. The low frequency of pyuria in patients from whom this organism was isolated is not surprising; it is well known that it does not always
produce a leucocyte response. The problem of apparently sterile pyuria is common in clinical and laboratory practice, and tuberculosis is only rarely found to be the cause. It is seen in patients with long-standing chronic pyelonephritis, long-standing cystitis, so-called interstitial cystitis, and prostatitis. In all these disorders the oxygen tension of the tissues of the urinary tract is likely to be lower than that of the urine itself, allowing multiplication of fastidious species of bacteria such as those we isolated. In our experience a search for such organisms in the urine of patients with apparently sterile pyuria is often rewarding. The protocol used in this study is practicable in a service laboratory and will detect streptococci that would otherwise be missed. The API identification of species not identifiable by standard subculture methods may be omitted and the isolates reported as fastidious streptococcus species. A few streptococci will be wrongly reported as coryneforms if API identification of catalase-negative coccobacilli is not undertaken. Incubation for up to 48 h in 7% carbon dioxide will detect about 800,, of the organisms without any extra expense; the remainder require additional culture media. Our practice is to ask clinicians to send repeat specimens, requesting culture on additional media, if patients have persistent symptoms or pyuria. We thank our laboratory colleagues for their cooperation, and Mrs T. Diplock for preparing the typescript. The study was supported by a grant from the Wessex Kidney Research Fund. Correspondence should be addressed to R. M. REFERENCES 1. Editorial. Streptococcus milleri, pathogen in various guises. Lancet 1985; ii: 1403-04. 2. Brumfitt W, Gargan RA, Hamilton-Miller JMT. Diagnosis and cure of recurrent urinary infection with microaerophilic and anaerobic bacteria. Br Med J 1980; 281: 909-10.
3. Reid RI, Webster O, Pead PJ, Maskell R. Comparison or urine bag-changing regimens in elderly catheterised patients. Lancet 1982; ii: 754-56. 4 Emmerson AM, Eykyn S. Streptococcus mutans endocarditis—a trap for the unwary. Br Med J1977, i: 905. 5 Clarke M, Pead L, Maskell R. Urinary infection in adult men: a laboratory perspective. Br J Urol 1985; 57: 222-26. 6. Parker MT, Stringer J The pattern of systemic disease due to Group-B streptococci. In Parker MT, ed. Pathogenic streptococci. Chertsey: Reedbooks, 1979. 171. 7. Abercrombie GF, Allen J, Maskell R. Corynebacterium vaginale urinary tract infection in a young man. Lancet 1978; i: 766.
TRANSMISSION OF CHRONIC IDIOPATHIC VITRITIS IN MICE BY INOCULATION OF HUMAN VITREOUS CONTAINING LEUCOCYTE PHAGOLYSOSOMAL BACTERIA-LIKE BODIES
EMIL WIROSTKO1
LEWIS A.
JOHNSON2
BARBARA M. WIROSTKO1
Edward S. Harkness Eye Institute1 and Department of Pathology,2 Columbia-Presbyterian Medical Center, New York, NY 10032, USA
Vitreous humour from chronic idiopathic vitritis (CIV) patients containing 0·5-0·7 µm diameter bacteria-like bodies (BLB) in polymorphonuclear leucocytes was inoculated into the eyelids of 100 mice. 200 control mice received either eyebank vitreous or saline. After 12 months, 53 mice that received CIV vitreous, but none of the controls, had clinical signs of ocular inflammation (p < 0·05); 15 of the mice that received CIV vitreous and none of the controls had histological evidence of chronic deep ocular inflammation, including vitritis (p < 0·05); 95 CIV-vitreous-inoculated and 38 control mice were dead (p < 0·05), and 3/3 of the CIV-vitreous group, compared with 0/3 controls, that were killed for histological assessment had phagolysosomal BLB identical to those in the CIV-vitreous inocula. The findings indicate that the BLB are pathogenic for mice.
Summary
Introduction ALTHOUGH chronic vitritis1—vitreous inflammation accompanied by inflammation of posterior ocular structures and known also as pars planitis, uveitis, choroiditis, iridocyclitis, iritis, chorioretinitis, scleritis, and optic neuritis-may be associated with systemic diseases, mainly arthritis syndromes,2 multiple sclerosis,2inflammatory bowel diseasessarcoidosis/and psoriasis,3it is commonest in apparently healthy adults,2in whom the condition is thought to be idiopathic.2 No microbiological agent has been isolated but in patients with chronic idiopathic vitritis (CIV) electron-microscopy of the vitreous often shows noncultivatable 0-5-0-7 jjm coccal shaped bacteria-like bodies (BLB) in phagolysosomes of some of the polymorphonuclear leucocytes in a background of predominantly mononuclear leucocytes.4 We describe here our attempts to transmit the disease by transfer of the BLB.
Materials and Methods Vitreous samples.-CIV vitreous was obtained’ from the first four of eight previously described CIV patients whose specimens contained the BLB. The 4 CIV vitreous specimens were stored at 4°C until animal inoculation. Animal inoculations.-According to a protocol accepted by the Institutional Review Board, 300 CD/1 male mice, 12-16 weeks old, weighing 15-20 g, were inoculated on the same day. Each mouse received subcutaneous injections of 0- 1 ml of vitreous into the lateral part of each lower eyelid. Each CIV vitreous specimen was inoculated into 25 mice (total 100 mice). Vitreous from each of 10 eye-bank eyes was inoculated into 10 mice, and sterile saline was inoculated into 100 mice (total of 200 control mice).
vascular resistance are not mutually exclusive forms of renal impairment. The clinically observed reversible renal impairment may be more closely related to the latter, whereas previously reported irreversible renal damage in cyclosporin-treated patients21,22 may be evidence of longterm effects of tubular toxicity. The methods by which cyclosporin causes renal vasoconstriction are unknown. Nonetheless, Siegl et al14 believe that the vascular effect (as opposed to renal tubular effects) is the prime mechanism of the renal impairment. Animal studies have suggested a role for a cyclosporinstimulated renin-angiotensin system, 14,23 whereas human studies19.24 have suggested that the renin-angiotensin system is actually suppressed by cyclosporin. Our studies did not address this question, but mean arterial pressure was significantly higher in our prospective study patients before conversion from cyclosporin to azathioprine. Non-reninmediated post-transplantation hypertension is rare in our experience, and we believe the role of the renin-angiotensin system in cyclosporin-induced post-transplantation hypertension needs further study. It is apparent that the cyclosporin-induced rise in vascular resistance is reversible, when patients are converted to azathioprine after nearly a year of treatment. It is not certain, however, whether longer exposure to cyclosporin might lead to permanent damage to the allograft. Prolonged renal vasoconstriction could cause tubular damage and eventual fibrosis and chronic nephron loss. This effect would be difficult to establish in kidney transplant patients, since chronic rejection could also cause chronic nephron loss. Even among large cardiac transplant centres the evidence that cyclosporin causes chronic irreversible renal damage is debated.25 A better understanding of the way cyclosporin causes renal vasoconstriction might reveal other, perhaps pharmocological, manipulations that could reverse its haemodynamic effect. It might also be possible to use a reduced dose regimen of cyclosporin that maintained the immunosuppressive effects but did not raise renal vacular resistance. If the renal vasoconstrictive effects of cyclosporin are, indeed, dose related the trend for some centres to use triple therapy with prednisone, low-dose cyclosporin, and low-dose azathioprine regimens may gain further support. This study was supported by a grant from the General Clinical Research Center (RR-0032) National Institutes of Health.
Corespondence should be addressed to J. J. C., Univesity of Alabama at Birmingham, Nephrology Research and Training Center, Birmingham, Alabama 35294, USA. REFERENCES 1. The Canadian Multicentre Transplant Study Group. A randomized clinical trial of cyclosporine in cadaveric renal transplantation. N EnglJ Med 1983; 309: 809-12. 2. European Multicentre Trial Group. Cyclosporin in cadaveric renal transplantation: one year follow-up of a multicentre trial. Lancet 1983; i: 986-90. 3. Bennett WM, Pulliam JP. Cyclosporine nephrotoxicity. Ann Intern Med 1983; 305: 267-73. 4. Chapman JR, Griffiths D, Harding NG, Morris PJ. Reversibility of cyclosporin nephrotoxicity after three months’ treatment. Lancet 1985; i: 128-30. 5. Flechner SM. Cyclosporin therapy. Transplant Immunol 1984; 1: 1-4. 6. Tauxe WN, Dubovsky EV, Kidd T. New formulae for the calculation of effective renal plasma flow. Eur J Nucl Med 1982; 7: 51-54. 7. Dubovsky EV, Russell CD. Quantitation of renal function with glomerular and tubular agents Semin Nucl Med 1982; 12: 308-29. 8. Lustig S, Silis V, Berger ME, et al. Effect of low dose cyclosporin A on blood pressure and arterial contractility in spontaneously hypertensive rats (abstract). American Society of Nephrology, New Orleans. December 15-18, 1985: 102A. 9. Moss NG, Powell SL, Falk RJ. Intravenous cyclosporine activates afferent and efferent renal nerves and causes sodium retention in innervated kidneys in rats. Proc Natl Acad Sci USA 1985; 82: 8222-26. 10. Gazdar AF, Dammin GJ. Neural degeneration and regeneration in human renal transplants. N Engl JMed 1979; 283: 222-24.
STREPTOCOCCI AS URINARY PATHOGENS LYNN E. COLLINS ROSEMARY W. CLARKE ROSALIND MASKELL Public Health Laboratory, St Mary’s Hospital, Portsmouth PO3 6AQ
In a 2-month prospective study of streptococci isolated from urine specimens in the laboratory, 242 strains of catalase-negative gram-positive cocci or coccobacilli were isolated in substantial numbers from 11 725 specimens. These comprised 10% of the important isolates. Species identification of all isolates was undertaken. 74 (30%) of the isolates were of species other than Streptococcus faecalis and S agalactiae. 79 (33%) were not detected on cysteine-lactose-electrolyte-deficient agar after overnight incubation in a carbon dioxide incubator. 20 of the 24 isolates of coccobacilli were Gardnerella vaginalis. Many of the isolates of fastidious species were accompanied by pyuria. An isolation protocol practicable in busy laboratories is proposed.
Summary
Introduction OVERNIGHT aerobic culture of urine specimens on primary isolation media leaves the symptoms of many patients and much pyuria unexplained. There has been interest in the pathogenicity of the fastidious species of streptococcil and there have been occasional reports of their isolation from midstream urine samples of patients with urinary-tract infection2 and from bladder urine of catheterised patients.3 We have undertaken a prospective study of streptococcal urinary infection, using methods capable of detecting such organisms. Some fastidious streptococci can appear coryneform (coccobacillary) in gram-stained preparations made from cultures on solid media;4 so that such species should not be missed we included all catalasenegative gram-positive cocci and coccobacilli in the study. Dieperink H, Starklint H, Leyssac PP. Nephrotoxicity of cyclosporine—an animal model: study of the nephrotoxic effect of cyclosporine on overall renal tubular function in conscious rats. Transplant Proc 1983; 15 (suppl 1): 2736-41. 12. Humes HD, Jackson NM, O’Connor RP, Hunt DA, White MD. Pathogenetic mechanisms of nephrotoxicity: insights into cyclosporine nephrotoxicity. Transplant Proc 1985; 17: 51-62. 13. Finn WF, Gitelman HG. N-acetyl-B-glucosaminidase excretion during cyclosporin A 11.
Human Toxicol 1984; 3: 416. et al. Cyclosporine, the renin-angiotensin-aldosterone system, and renal adverse reactions. Transplant Proc 1983; 15 (suppl 1): 2719-25. 15. McKenzie N, Devineni R, Vezina W, Keown P, Stiller C. The effect of cyclosporine on organ blood flow. Transplant Proc 1985; 17: 1973-75. 16. Paller MS, Murray BM, Ferris TF. Decreased renal blood flow after cyclosporine infusion (abstract). American Society of Nephrology, Washington, DC. Dec 9-12, 1984: 248A. 17. Jao S, Waltzer W, Arveit LA. Acute cyclosporin induced decrease in GFR is mediated by changes in renal blood flow and renal vascular resistance (abstract). American Society of Nephrology, New Orleans, Dec 1985: 282A. 18. Sullivan BA, Hak LJ, Finn WF. Cyclosporine nephtrotoxicity: studies in laboratory animals. Transplant Proc 1985, 17: 1145-54. 19. Whiting PH, Simpson JG, Davidson RJ, Thomson AW. Pathological changes in rats receiving cyclosporin A at immunotherapeutic dosage for 7 weeks. Br J Exp Pathol 1983; 64: 437-44. 20. Gerkins JF, Bhagwandeen SB, Dosen PJ, Smith AJ. The effect of salt intake on cyclosporine-induced impairment of renal function in rats. Transplantation 1983; 38: 412-17. 21. Myers BD, Ross J, Newton L, Luetscher J, Perlroth M. Cyclosporine associated chronic nephropathy. N Engl J Med 1984; 311: 699-705. 22. Rao VK, Crosson JT, Kjellstrand CM. Chronic irreversible nephrotoxicity from treatment.
14.
Siegl H, Ryffel B, Petric R,
23.
cyclosporin A. Nephron 1986; 41: 75-77 Siegl H, Ryffel B. Effect of cydosporin on renin-angiotensin-aldosterone system. Lancet 1982; ii: 1274.
Bantle JP, Nath KA, Sutherland DER, Najarian JS, Ferris TF. Effects of cyclosporine on the renin-angiotensin-aldosterone system and potassium excretion in renal transplant recipients. Arch Intern Med 1985; 145: 505-08. 25. Porter G Discussion IV, diagnosis, incidence, and severity of cyclosporine nephrotoxicity. Transplant Proc 1985; 17: 232-44. 24.
480 TABLE II—CULTURE MEDIUM AND LENGTH OF INCUBATION
Methods Between Jan 1 and Feb 28,1986, we identified all the streptococci isolated from urine cultures on which sensitivity tests were carried out. The decision as to the importance of each isolate, and therefore whether to carry out sensitivity tests, was made in the same way as for all other urinary isolates. Factors taken into consideration included bacterial count, purity of growth, presence of pyuria (defmed as 10 cells/1 uncentrifuged urine), and relevant clinical details. Uncentrifuged urine was examined by the inverted microscope method. All specimens were inoculated onto cysteine-lactoseelectrolyte-deficient (CLED) agar by means of a 0-005 ml standard loop. Cultures were incubated overnight at 37°C in an atmosphere containing 7°o carbon dioxide. Any cultures that left either symptoms or pyuria unexplained after overnight incubation were reincubated for a further 24 h. Additional culture procedures were carried out on repeat specimens for which culture for fastidious organisms had been requested (usually because of a finding of apparently sterile pyuria on the previous specimen) and on specimens from patients with symptoms of prostatitis or epididymo-orchitis, in which fastidious organisms have been implicated.5 These specimens were also inoculated onto chocolated blood agar (CBA) and blood agar containing 16 J.lg/ml nalidixic acid (BAN). The CBA plate was incubated in 7% carbon dioxide and read at 18 and 48 h. The BAN plate was incubated for 48 h in an anaerobic cabinet (Don Whitley Mk II). All isolates of gram-positive, catalase-negative cocci or coccobacilli were identified by standard laboratory procedures-mannite fermentation on electrolyte-deficient agar (Streptococcus faecalis), orange coloration on Islam’s medium (S agalactiae), a strong caramel smell associated with sensitivity to penicillin (S milleri), growth anaerobically only with sensitivity to metronidazole (anaerobic streptococcus). Any organism not identified by these procedures was identified by means of API 20 STREP.
TABLE III-0RGANISMS ISOLATED FROM
30 SPECIMENS SHOWING
APPARENTLY STERILE PYURIA AFTER’OVERNIGHT INCUBATION ON CLED AGAR
specimens. 55% of the specimens showed pyuria, with the highest incidence (76%) in women of over 55 years. 24 isolates were coryneform on gram-stained film: 20 of these, including 3 from male patients, were Gardnerella vaginalis ; 3 S morbillorum; and 1 was a coryneform organism. Table II shows the numbers of isolates detected on CLED overnight incubation or after 48 h incubation or detected only on CBA and/or BAN plates. 30 specimens showed apparently sterile pyuria after overnight incubation on CLED agar. Table III shows the organisms subsequently isolated and the age and sex distributions of the patients from whom the specimens came.
agar after
Results
During the study period 11 725 urine specimens were examined. 2486 (21 %) isolates from these specimens were considered to be important, and of these 242 (10%) were included in the study. Table I shows the age and sex distributions of the patients from whom they were isolated. There were 71 isolates from 65 male patients and 171 isolates from 166 female patients. 81 (35%) of the patients were in hospital. 1 isolate was from a suprapubic aspirate, 17 from catheter specimens, and the remainder from midstream
Discussion
Although the duration of this study was short, the numbers of specimens examined and organisms isolated were large and some conclusions can reasonably be drawn. S faecalis and S agalactiae (Lancefield group B) are well-recognised urinary pathogens. 6 (5%) isolates of S faecalis and 11 (20%) of S agalactiae would not have been
TABLE I-ISOLATES OF CATALASE-NEGATIVE GRAM-POSITIVE COCCI AND COCCOBACILLI FROM URINE
Figures in parentheses number of specimens with pyuria. unidentifiable by the techniques used. =
*Isolated from
a
suprapubic aspirate.
tIsolated is mixed culture with S millen. tIsolates which were
481
detected after overnight incubation of cultures on CLED agar. 1 isolate of each organism grew only on additional media. The detection rates of the more fastidious species after overnight incubation of CLED agar cultures were much lower; 17 (74%) of isolates of S milleri, 25 (81%) of other streptococci, and 20 (100%) of G vaginalis required longer incubation. 43 (58%) of the 74 isolates of these species grew only on the additional culture media. It is not possible to say whether the detection rate would have been lower still if overnight incubation in air had been used, as it is for urine culture in most laboratories. However, it is very likely, at least, that the 12 isolates of S milleri and other fastidious streptococci that were detected after overnight incubation would have been missed. 3 isolates of streptococci, all S morbillorum, would have been missed if catalase-negative coccobacilli had not been included. It is interesting that both S agalactiae and G vaginalis were isolated from male patients; both are known to be 6 present in the female genital tract. Parker and Stringer pointed out that systemic infections with S agalactiae affect males as much as females, and Abercrombie and colleagues7 reported the isolation of G vaginalis from the bladder urine of a young man with acute cystitis. The low frequency of pyuria in patients from whom this organism was isolated is not surprising; it is well known that it does not always
produce a leucocyte response. The problem of apparently sterile pyuria is common in clinical and laboratory practice, and tuberculosis is only rarely found to be the cause. It is seen in patients with long-standing chronic pyelonephritis, long-standing cystitis, so-called interstitial cystitis, and prostatitis. In all these disorders the oxygen tension of the tissues of the urinary tract is likely to be lower than that of the urine itself, allowing multiplication of fastidious species of bacteria such as those we isolated. In our experience a search for such organisms in the urine of patients with apparently sterile pyuria is often rewarding. The protocol used in this study is practicable in a service laboratory and will detect streptococci that would otherwise be missed. The API identification of species not identifiable by standard subculture methods may be omitted and the isolates reported as fastidious streptococcus species. A few streptococci will be wrongly reported as coryneforms if API identification of catalase-negative coccobacilli is not undertaken. Incubation for up to 48 h in 7% carbon dioxide will detect about 800,, of the organisms without any extra expense; the remainder require additional culture media. Our practice is to ask clinicians to send repeat specimens, requesting culture on additional media, if patients have persistent symptoms or pyuria. We thank our laboratory colleagues for their cooperation, and Mrs T. Diplock for preparing the typescript. The study was supported by a grant from the Wessex Kidney Research Fund. Correspondence should be addressed to R. M. REFERENCES 1. Editorial. Streptococcus milleri, pathogen in various guises. Lancet 1985; ii: 1403-04. 2. Brumfitt W, Gargan RA, Hamilton-Miller JMT. Diagnosis and cure of recurrent urinary infection with microaerophilic and anaerobic bacteria. Br Med J 1980; 281: 909-10.
3. Reid RI, Webster O, Pead PJ, Maskell R. Comparison or urine bag-changing regimens in elderly catheterised patients. Lancet 1982; ii: 754-56. 4 Emmerson AM, Eykyn S. Streptococcus mutans endocarditis—a trap for the unwary. Br Med J1977, i: 905. 5 Clarke M, Pead L, Maskell R. Urinary infection in adult men: a laboratory perspective. Br J Urol 1985; 57: 222-26. 6. Parker MT, Stringer J The pattern of systemic disease due to Group-B streptococci. In Parker MT, ed. Pathogenic streptococci. Chertsey: Reedbooks, 1979. 171. 7. Abercrombie GF, Allen J, Maskell R. Corynebacterium vaginale urinary tract infection in a young man. Lancet 1978; i: 766.
TRANSMISSION OF CHRONIC IDIOPATHIC VITRITIS IN MICE BY INOCULATION OF HUMAN VITREOUS CONTAINING LEUCOCYTE PHAGOLYSOSOMAL BACTERIA-LIKE BODIES
EMIL WIROSTKO1
LEWIS A.
JOHNSON2
BARBARA M. WIROSTKO1
Edward S. Harkness Eye Institute1 and Department of Pathology,2 Columbia-Presbyterian Medical Center, New York, NY 10032, USA
Vitreous humour from chronic idiopathic vitritis (CIV) patients containing 0·5-0·7 µm diameter bacteria-like bodies (BLB) in polymorphonuclear leucocytes was inoculated into the eyelids of 100 mice. 200 control mice received either eyebank vitreous or saline. After 12 months, 53 mice that received CIV vitreous, but none of the controls, had clinical signs of ocular inflammation (p < 0·05); 15 of the mice that received CIV vitreous and none of the controls had histological evidence of chronic deep ocular inflammation, including vitritis (p < 0·05); 95 CIV-vitreous-inoculated and 38 control mice were dead (p < 0·05), and 3/3 of the CIV-vitreous group, compared with 0/3 controls, that were killed for histological assessment had phagolysosomal BLB identical to those in the CIV-vitreous inocula. The findings indicate that the BLB are pathogenic for mice.
Summary
Introduction ALTHOUGH chronic vitritis1—vitreous inflammation accompanied by inflammation of posterior ocular structures and known also as pars planitis, uveitis, choroiditis, iridocyclitis, iritis, chorioretinitis, scleritis, and optic neuritis-may be associated with systemic diseases, mainly arthritis syndromes,2 multiple sclerosis,2inflammatory bowel diseasessarcoidosis/and psoriasis,3it is commonest in apparently healthy adults,2in whom the condition is thought to be idiopathic.2 No microbiological agent has been isolated but in patients with chronic idiopathic vitritis (CIV) electron-microscopy of the vitreous often shows noncultivatable 0-5-0-7 jjm coccal shaped bacteria-like bodies (BLB) in phagolysosomes of some of the polymorphonuclear leucocytes in a background of predominantly mononuclear leucocytes.4 We describe here our attempts to transmit the disease by transfer of the BLB.
Materials and Methods Vitreous samples.-CIV vitreous was obtained’ from the first four of eight previously described CIV patients whose specimens contained the BLB. The 4 CIV vitreous specimens were stored at 4°C until animal inoculation. Animal inoculations.-According to a protocol accepted by the Institutional Review Board, 300 CD/1 male mice, 12-16 weeks old, weighing 15-20 g, were inoculated on the same day. Each mouse received subcutaneous injections of 0- 1 ml of vitreous into the lateral part of each lower eyelid. Each CIV vitreous specimen was inoculated into 25 mice (total 100 mice). Vitreous from each of 10 eye-bank eyes was inoculated into 10 mice, and sterile saline was inoculated into 100 mice (total of 200 control mice).