UJMS 110 (3) bra Upsala J Med Sci 110 (3): 217–231, 2005 Acute Glomerulonephritis Associated with Streptococcus Pyogenes with Concomitant Spread of Streptococcus Constellatus in Four Rural Families Almroth G1, Lindell Å1 , Åselius H1, Sörén L2, Svensson L3, Hultman P4, Eribe ERK5, Olsen I5. 1Departments of Nephrology and 2Clinical Microbiology, University Hospital of Linköping, 3Department of Internal Medicine, Hospital of Eksjö, 4Department of Molecular and Immunological Pathology, Linköping University, Sweden and 5Department of Oral Biology, Dental Faculty, University of Oslo, Norway. ABSTRACT We studied history, renal histopathology and microbiology of an epidemic of acute glomerulonephritis associated with throat infections and uncommon culture results in four neighbour families. A 40-year-old man (index patient) was referred to a university hospital for dialy- sis and kidney biopsy due to a suspected acute glomerulonephritis. An acute tonsil- litis had preceded the condition. Penicillin treatment had been started four days before the discovery of renal failure. Throat swabs were positive for �-hemolytic streptococci, group C (GCS). GCS were also found in throat cultures from his wife and two of their children. The bacteria were typed as Streptococcus constellatus. A third child had S. constellatus expressing Lancefield antigen group G. A neighbour and two of his children fell ill the following week with renal involvement. Throat swabs from both these children were positive for S. constellatus. His third child had erythema multiforme and S. constellatus in the throat while a fourth child had �- hemolytic streptococci group A; Streptococcus pyogenes. Kidney biopsies on the index patient and his neighbour showed an acute diffuse prolipherative glomeru- lonephritis compatible with acute post-streptococcal nephritis and microbiological analysis of renal tissue revealed in both cases S. pyogenes and S. constellatus. The Received 23 September 2004 Accepted 28 February 2005 Key words: glomerulonephritis, S. pyogenes, streptococci group C, streptococci group G, S. constellatus families had had much contact and had consumed unpasteurized milk from our index patient’s farm. In four of seven persons in two additional neighbouring fami- lies S. constellatus was found in throat swabs during the same month while two per- sons carried Streptococcus anginosus expressing the Lancefield C antigen. In conclusion spread of S. constellatus coincided with the occurrence of four cas- es of acute glomerulonephritis. The two biopsied patients had both S. pyogenes and S. constellatus present in renal tissue. The epidemic either suggested that the out- break of glomerulonephritis was due to S. pyogenes but coincided with the trans- mission and colonization of S. constellatus or that the S. constellatus strains were highly pathogenic or nephritogenic and that this organism can be transmitted in such cases. INTRODUCTION Streptococcus pyogenes or �-hemolytic streptococci group A (GAS) is recognized as the causative agent of tonsilllitis, impetigo, scarlet fever and septicemia and can induce glomerulonephritis and rheumatic fever as late complications [1, 2]. The relationship between acute glomerulonephritis and infections with GAS was first established in the early part of the 20th century, when Rammelkamp and co- workers showed that only certain strains of group A streptococci, especially type 12, were able to induce acute glomerulonephritis [3]. Cases or epidemics of acute glomerulonephritis are usually associated with pharyngeal infections or skin infec- tions, i.e. impetigo [3, 4]. �-Hemolytic streptococci group C (GCS) can cause tonsillitis but very rarely glomerulonephritis [5, 6]. Group G streptococci have been considered to be less pathogenic than group C streptococci but have been associated both with glomeru- lonephritis and renal failure [7, 8]. Streptococcus constellatus, belonging to the “milleri” or “anginosus” group of streptococci sometimes express the Lancefield C-antigen. These bacteria differ from the “large colony type” GCS (Streptococcus zooepidemicus) in forming small, “pin point” colonies and having a different pathogenic potential. They belong to the nor- mal flora of the mouth. They have not, to our knowledge, been associated with ton- sillitis or glomerulonephritis but have been shown to occur in pharyngitis and peri- tonsillitis [9–11]. In this report we describe an epidemic of acute glomerulonephritis coinciding with the spread of S. constellatus in the patients families and in neigh- bouring families in a rural area of Sweden. Possible associations of the spread of the S. constellatus and the occurrence of four cases of acute glomerulonephritis, two of them biopsy-verified and one of them with a nephrotic syndrome, are discussed. 218 PATIENTS AND METHODS Case reports: A 40-year old man-the index patient (A)-was admitted to his local hospital, Eksjö, with signs of acute nephritis. Due to a high grade of uremia he was transferred the following day to the University Hospital of Linköping, Sweden for kidney biopsy and acute dialysis. Four days before arrival, he had started on a course of penicillin due to acute tonsillitis. Three further patients (B-D) described below later developed clinical findings indicating an acute glomerulonephritis. The acute renal failure in patient A remitted after treatment, transiently with cyclophosphamide, corticosteroids, plasmapheresis and dialysis. A kidney biopsy showed acute postinfectious glomerulonephritis.The patient was discharged from the hospital with a serum creatinine of 173 �mol/l (normal range 70-115 �mol/l). At the time of discharge a throat culture was positive for beta-hemolytic streptococci, Lance- field group C, reported as GCS. The same type of bacteria were also detected in throat cultures from the patient´s wife and two of their children. A third child had �- hemolytic streptococci Lancefield group G (GGS) in a culture from the throat while a fourth child was culture-negative. Patient A´s renal function was subsequently normalized with both normal serum creatinine values and a normal iohexol clearance of 99 ml/min and 1.73 m2 body square area in November 1997. There was no proteinuria but a slight hematuria (++) . The week after the start of patient A´s illness a neighbour of our patient (B) fell ill (Figs 1 and 2). He was admitted to his local hospital, Eksjö, with renal failure (S- creatinine 200 �mol/l and urea 35 mmol/l, normal range 3-9 mmol/l). The neigh- bour had felt ill for about four weeks and had had a slight oedema. One week before 219 A Tonsillitis, penicillin nasopharynx culture neg. Glomerulonephritis GCS in throat culture 12/5 15/5 16/5 20/5 B Tonsillitis, penicillin , throat and nasoph. culture neg Glomerulonephritis GGS in throat culture 14/5-15/5 21/5 2/6 C Throat pain Glomerulonephritis-GCS in the throat April 20/5 D Glomerulonephritis GCS in the throat _________________________________________________________________________________________ 20/5 22/5 Fig 1. The time points of tonsillitis, throat cultures and glomerulonephritis in patients A–D. admittance he had received a course of penicillin. A throat culture before the course was negative. Antistreptolysin O titre (ASO) was 200 (normally < 150) and anti- deoxyribonuclease B (ADNase B) titre was high, >1200 (normally < 400). A kidney biopsy was performed, see histopathology below. A throat culture 12 days after- wards was positive for GGS. His wife was healthy. Two of the neighbour´s children (C and D) were admitted to the central hospital of Ryhov in Jönköping. Both had fallen ill with renal involvement. The boy (C) had earlier had a sore throat. He had a hypertension with blood pressures of 140/80- 160/120 mm Hg and was transiently treated with metoprolole. Creatinine was 71-83 �mol/l and urinary albumin 0.38 g/24 h (normally below 0.1 g/24h). Urinary sedi- ment findings were pathologic. ASO was elevated to 560 and ADNase was also 220 Family 1 (farmer family) M 9, F S.constellatus. F 15, G S.constellatus. (Lancefield Group C) M 14, H No symptoms S constellatus (Lancefield group C) M 8, I S.constellatus (Lancefield Group C) M 40, A Nephritis Streptococci C F 35, E Healthy S Constellatus Lancefield group C Family 2 M 10, C Nephritis S. constellatus (Lancefield group C) F 12, D Nephritis S. constellatus. (Lancefield group C) M 7, K Multiforme erythema S.constellatus (Lancefield group C) M 2, L Ill two weeks earlier S. pyogenes (Lancefield group A) . M 41, B Nephritis (S.constellatus 12 days later, Lancefield group G) F 35, J Healthy Family 3 M 8, O S.constellatus (Lancefield group C) M 6, P S.constellatus (Lancefield group C) F 2, Q S. constellatus (Lancefield group C) M 36, M S. anginosus (Lancefield group C) F 34, N S. anginosus (Lancefield group C) Family 4 M 30, R S. constellatus (Lancefield group C) F 28, S (S. constellatus 14 months later) Fig 2. Schematic drawing of family 1 (farmer family). Schematic drawing of family 2 (neighbours). Schematic drawings of families 3 and 4 (neighbours). high > 1200. Complement level C3 was low (0.14g/l), while C4 was normal (0.22 g/l). His twelve year old sister (D) had a nephrotic syndrome with urinary albumin levels of 3.3-9.7 g/l (normal < 0.03 g/l) and a serum albumin of 29-32 g/l (normal > 35 g/l). Serum creatinine was 69-98 �mol/l and the blood pressure was elevated to 150/90-150/100 mm Hg. In both children beta hemolytic streptococci, reported as GCS, were found in throat cultures. A third child who acquired erythema multi- forme also had the same bacteria in the throat, while a forth child had group A strep- tococci (Streptococcus pyogenes). The time points of tonsillitis, throat cultures and onset of glomerulonephritis are shown in Fig. 1. The families had had multiple contacts, and their children usually went to school by the same bus. The patient who first fell ill was a farmer, and the families had consumed unpasteurized milk from the farm of patient A. Cultures from the milk were, however, negative for GCS and other streptococci. In six of seven persons of two further neighbouring families, ß-hemolytic streptococci, reported as GCS, were found in throat swabs during the same month. Five of these persons were treated with penicillin. A throat culure from one of these patients 14 months later revealed beta-hemolytic streptococci group C. Microbiology: Throat swabs. Throat swabs were transported in Amies medium and cultured on 5% sheep blood agar containing Columbia agar base (Difco). �-hemolytic colonies 221 Fig 3. Pulse field gel electrophoresis of streptococci. Lane 1: DNA marker � ladder. Lanes 2-16: pati- ents DNA. Lane 17: DNA marker S. aureus 8325 (NCTC 8523). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1. DNA marker λ ladder 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. DNA marker S. aureus 8325 Fig 2 Pulse field gel electrophoresis of streptococci were isolated and agglutinated with latex particles coated with antibodies to Lance- field group A, C and G antigens (Oxoid Ltd, Basingstoke, Hampshire, England). Pulsed Field Gel Electrophoresis (PFGE). Chromosomal DNA from GBS embed- ded in agarose plugs was prepared as previously described [12]. An agarose slice was incubated overnight at 30°C with 20 U of SmaI, (MBI, Fermentas) in the pro- vided enzyme buffer. Plugs were washed in TE-buffer (1h 5°C) before mounting into wells of a 1 % agarose gel (Pulsed Field Certified Agarose, Bio Rad) in 0.5x TBE-buffer pH 8.3. Electrophoresis was performed on an automated PFGE appara- tus (Gene Path strain typing system, Bio Rad). Standard programs for fragment size 50-600 was used. A standard �-ladder (New England Biolabs Inc, MA, USA) and SmaI digested S. aureus 8325 was included alongside the samples. The agarose gel was stained with 0.2% ethidiumbromide and washed in tap water visualized and photographed under UV light (Fig. 3). PCR analysis of skaA Five to ten colonies, picked from a blood agar plate, were resuspended in 50 ul ster- ile distilled water in a micro test tube and heated for 5 min at 100°C. After centrifu- gation, 10 ul of the supernatant was used as template in the PCR reactions. Reaction conditions were 0.2 mm dNTP´s, 20pmol of each primer with 0.5 U Taq polymeras- es buffer with 1.5 mM MgCl 2 (MBI Fermenta, Labassco) and bovine serum albu- min (0.17 mg/ml). The amplification was performed on a PTC-200 thermal cycler (MJ Research, Scandinavian Diagnostic Services, (SDS) Falkenberg, Sweden) with annealing temperature of 55°C. Forward primer used was ska 453-477; 5´-AAC- CTTGCCGACCCAACCT-3´ and reverse primer ska 770-791; 5´-TCCATTG- GTAAAATCGTACGG-3´ [13]. The PCR products were separated with elec- trophoresis on 0.7 % agarose gel. Phenotypic typing of group C streptococci Further typing of group C streptococci was performed with API Strep from Bio- Mérieux sa Marcy l´Étoile, France. Renal biopsy examination Part of the renal biopsy material obtained from patients A and B was snap-frozen in isopentane-CO2 for immunfluorescence studies. Cryostate sections, 4-5 �m thick, were fixed in ethanol, incubated with FITC-conjugated rabbit antibodies to human IgG, IgA, IgM, and C3c (Dakopatts, Sweden), and examined in an incident-light fluo- rescence microscope. Another part of the biopsy material was immersed in Histo- choice (Amresco, Solon, IL, USA), embedded in paraplast, and cut into 1- to 2-�m sections that were stained with periodic acid Schiff´ s reagent and with periodic acid silver methenamine. The remaining part of the biopsies was immersed in 2-% glu- taraldehyde, embedded in Epon 812. Semi- and ultra-thin sections were prepared, stained with OsO4 and examined in a JEOL 1200 electron microscope. 222 223 Table 1: Pulse field gel electrophoresis - description of lanes in fig. 2. Lane Bacteria Lancefield group Patient 2 S. constellatus G B 3 " G F 4 S. constellatus C D 5 " C C 6 " C K 7 S.anginosus C M 8 " C N 9 S.constellatus C O 10 " C P 11 " C Q 12 " C R 13 " * C S 14 " C H 15 " C G 16 " C I 1 DNA marker, λ-ladder 17 DNA-marker, S. aureus 8325 *) isolated 14 months after the outbreak Table 1: Pulse field gel electrophoresis – description of lanes in Fig 3. Tissue preparation and lysis A Nucleon kit [14] for the extraction of genomic DNA from tissue (Nucleon HT, product code RPN 8509), Amersham International plc & Scotlab Ltd 1997) was used. The tissue biopsies were grinded on liquid nitrogen to fine powder in a sterile mortar. Grinding occurred in a laminar air hood. Grinded tissue was transferred to an Eppendorf tube and 350 �l Reagent B was added. Thereafter 5 �l RNase solution was added to the tube and the contents were incubated for 30 min at 37°C. Pro- teinase K solution (18 �l) was added and the tissue was incubated at 56°C overnight. After centrifugation at 7,500 rpm for 5 min the supernatant was trans- ferred to a new tube. For deproteinization 100 �l of sodium perchlorate solution was added and the content mixed by inverting the tube several times. DNA extraction Chloroform (600 �l) was added to the supernatant and the content mixed by invert- ing the Eppendorf tube several times. Nucleon resin (150 �l) was then added with- out mixing the content. Centrifugation occurred at 2,200 rpm for 1 min. The super- natant was transferred to a new tube and DNA was precipitated by adding 2 vol- umes (700 �l) of cold ethanol and by inverting the tube. The DNA was washed by centrifugation at 4,000 x g for 5 min. The supernatant was discarded and 1000 �l of 70% cold ethanol was added. After mixing, another high-speed centrifugation was made and the supernatant was discarded. The DNA pellet was dried in a vacuum evaporator for 15 min. Thereafter, the DNA was dissolved in TE buffer. Checkerboard DNA-DNA hybridization A slightly modified procedure of the checkerboard method developed by Socransky et al. [15] was used for DNA-DNA hybridization. Each DNA sample was pipetted in volumes of 100 �l and transferred to separate Eppendorf tubes. One hundred microliter of 0.5 M sodium hydroxide was added to the sample. All samples were placed in a water bath (100°C) and boiled for 5 min. They were then neutralized using 800 �l of 5 M ammonium acetate. The released DNAs from the clinical species and the type strains of S. constellatus NCDO 2226T and S. pyogenes CCUG 4207T were placed into the lanes of a Minislot (Immunetics, Cambridge, MA, USA) and then deposited on a nylon membrane (Boehringer Mannheim, Mannheim, Ger- many). The membranes were fixed by baking at 68°C for 30 min followed by expo- sure to ultraviolet light for 30 s. The membrane with the fixed DNA was placed in a Miniblotter 45 (Immunetics) with the lanes of DNA at 90°C to the channels of the device. The membranes were prehybridized at 42°C for 1 h in 50% formamide (AppliChem, Darmstadt, Germany), 5 x SSC (1 x SSC = 150 mM sodium chloride, 15 mM sodium citrate (AppliChem), pH 7.0), 10% casein (Sigma, St. Louis, MO, USA), 5 x Denhardt’s reagent (ApplicChem), 25 mM sodium phosphate (pH 6.5) and 10 mg/ml yeast RNA (Boehringer Mannheim). Digoxigenin-labeled, whole genomic probes and hybridization buffer containing 45% formamide (5 x SSC, 1 x Denhardt’s reagent, 20 mM sodium phosphate (pH 6.5), and 10 mg/ml yeast RNA, 224 20 ng/ml labelled probe, 10% dextran sulphate (Sigma) and 10% casein were placed in individual lanes of the Miniblotter. The whole apparatus was covered with Saran wrap and transferred to a sealed plastic dish with water. The membranes were hybridized overnight with gentle shaking at 42°C. They were washed in a plastic dish to remove loosely bound probe. To detect hybrids, the membranes were blocked with 10% casein in maleate buffer (100 mM maleic acid, 150 mM sodium chloride, pH 7.5) and then incubated with antidioxigenin antibody conjugated with alkaline phosphatase (Boehringer Mannheim), diluted 1:20 000 in maleic buffer. Signals were detected chromogenically using NTB/BCIP tablets (Boehringer Mann- heim) overnight. RESULTS Microbiology PFGE typing showed that 13/15 isolates reported as GCS were in fact S. constella- tus and 2/15 were S. anginosus (Table 1 and Fig 3). Reagglutination confirmed that 13 of these 15 strains expressed the Lancefield C-antigen and that two belonged to group G. Eleven of the 13 S.constellatus strains expressed the C antigen. PFGE also showed that 10 of the 11 S. constellatus group C were identical (Fig 3, lane 4, 5, 6, 9, 10, 11, 12, 14, 15, 16). The eleventh strain of S. constellatus (Fig 3, lane 13) obtained from a throat culture 14 months after the outbreak, differed somewhat from the other 10. PCR-analysis of the ska gene showed that this gene was absent in all 15 isolates. The group C strain from the index patient (A) was not subtyped as it had been isolated in a different laboratory (Linköping) and was therefore not avail- able for further analysis. Epidemiology The time points of tonsillitis and throat cultures are shown in Fig. 1. The family trees and the results of the throat cultures with subtyping are shown in Fig 2. Kidney histopathology The kidney biopsy material from both patients A and B contained renal cortex with approximately 20 glomeruli. Histologically, a moderate to severe increase of glome- rular endocapillary cells in a diffuse and global pattern was observed. The proliferating endocapillary cells, together with infiltrating polymorphonu- clear cells,encroached considerably on the capillary lumina (fig. 4a). In patient B occasional capillary loops contained a hyaline subendothelial material. There was no significant extra-capillary cell proliferation. Examination by immunfluorescence microscopy revealed in patient A large amounts of IgG and C3c, and moderate amounts of C1 q, outlining the glomerular capillary loops in a granular, "starry sky" pattern. Slight amounts of granular IgM and IgA deposits were also seen along the 225 capillary loops. Patient B showed the same pattern of granular deposits with a dom- inance of C3c along the capillary loops (Fig. 4b), moderate amounts of IgG, and slight amounts of IgA, while there were no deposits of IgM and C1q. Both patients showed granular deposits of immunoglobulins and complement in the mesangial region. The ultrastructural examination confirmed presence of endocapillary cell hyperplasia, and electrone-dense subendothelial deposits. In some capillary loops in patient B, these electron-dense subendothelial deposits filled a large part of the cap- 226 Fig 4a. Irregular deposits of C3c along the glomerular capillary loops and in the mesangial regions, so-cal- led “starry sky pattern”. Fig 4b. Glomerulus with accentuated lobular configuration due to severe endocapillary cell proliferation. The proliferating cells together with infiltrating polymorphonuclear leukocytes fill out the capillary lumina. illary lumen. Both patients had discrete, large, irregular subepithelial electron-dense deposits, so called “humps” (Fig. 4c). Patient A showed foci of tubulointerstitial in- flammation sometimes with tubulitis. Renal tissue analysis The kidney biopsies from patients A and B and one of the 6 controls, a patient with IgA nephropathy, were positive for S. pyogenes. Checkerboard DNA-DNA hybridization showed that both renal biopsies con- tained S. constellatus. S. constellatus was positive for the control patients PL 1723-98, PL 10496-98 and PL 12992-98 but negative for the patients PL 400-98 and PL 12732-98. The latter five control-patients were negative for S. pyogenes. Their renal tissue contained, according to light microscopy, mainly nephrosclerosis. DISCUSSION This study describes an outbreak of glomerulonephritis associated with the isolation of S. pyogenes in two kidney biopsies, but also with identical strains of S. constellatus in throat cultures, and in both cases in renal biopsies, from the patients and members of the families and other families in the neighbourhood (Fig. 2). S. constellatus was also present in 3/6 control kidney biopsies. S. constellatus belongs to the normal flora of the 227 Fig 4c. Large, discrete electron-dense deposits on the subepithelial side of the glomerular basement membra- ne, so-called “hump”, covered by a glomerular epithelial cell with many endocytic vacuoles. mouth from which it may spread. In the index patient (A) GCS were isolated but sub- typing was not performed. S. pyogenes was positive also in a kidney biopsy of one control patient with chronic glomerulonephritis (i.e. IgA nephropathy). The remaining 5 specimens were negative for S. pyogenes. S. constellatus has also been found in abscesses and in peritonsillitis and in rare cases in association with pharyngitis [9–11]. The index patient (A) had a serious dialysis-requiring condition and a second patient (B) had a notable renal failure with a serum creatinine of 200 �mol/l. One child had glomerulonephritis with a nephrotic syndrome and another had glomeru- lonephritis (C, D). S. pyogenes may have been the main cause of glomerulonephritis in both of the kidney-biopsied patients. Whether S. constellatus was a concomitant cause of glomerulonephritis cannot be determined at present. Other bacteria than group A streptococci have increasingly been recognized as causes of acute post- infectious glomerulonephritis [16, 17]. An association between S. constellatus and glomerulonephritis has so far not been reported. The possibility of an infection with GAS or large colony type GCS, preceding the glomerulonephritis in the index patient and the three patients colonized with S. constellatus must be considered (Figs 4 a-b) even if throat cultures at the onset of tonsillitis were negative. This pos- sibility is corroborated by the finding of S. pyogenes in the renal tissue of two patients. This possibility might also be supported by the increased levels of AST and anti-DN:ase B in two patients suggesting a recent infection with GAS or possi- bly GCS [18–20]. S. constellatus is not, to our knowledge, known to produce strep- tolysin O or DN:ase B. Glomerulonephritis associated with large colony type GCS (S. zooepidemicus) has been described but is uncommon [5,6]. The mechanism is not known but may be the same as that proposed for glomerulonephritis associated with GAS, i.e. nephritogenic proteins (endostreptosin) produced by the bacteria [21]. Both of our kidney biopsied patients had a picture of acute diffuse proliferative glomeru- lonephritis of the kind seen in acute poststreptococcal glomerulonephritis. In acute post-streptococcal glomerulonephritis, NSAP (nephritogenic associated protein) with production of streptokinase from the streptococci has been suggested to induce the binding of complement to the glomeruli by itself and by a specific antibody [22–24]. If S. constellatus caused the glomerulonephritis such a mechanism seems unlikely, since the ska gene was not present in the S. constellatus strains isolated here. The mechanisms of acute post-streptococcal glomerulonephritis are however still unclear [3, 13]. Recently a bacterial protein H, a surface protein of S. pyogenes has been dis- cussed in the pathogenesis of acute poststreptococcal glomerulonephritis [25]. Circulating immune complexes with deposition and induction of complement and inflammatory cells in glomeruli as well as in situ antigen-antibody reactions are considered as the main mechanism of postinfectious glomerulonephritis, and bacte- rial antigens not present in GAS-induced nephritis may have been involved [3, 13]. 228 S. constellatus is not known to induce tonsillitis, but may occur in peritonsillar abscesses and periapical endodontic lesions of asymptomatic teeth [26, 27]. It may also be present in empyema thoracis and lung abscess and has been described in association with endocarditis [28, 29]. Glomerulonephritis has been described also in association with endocarditis caused by �-hemolytic (viridans) streptococci [30–32]. Such streptococci are however more able to induce chemotaxis of leuco- cytes than the streptococci belonging to the milleri group in which S. constellatus is included [33]. The presence of S. pyogenes in the biopsy of the control patient with IgA nephropathy may be coincidental. A connection between IgA nephropathy and post-streptococcal glomerulonephritis in some cases is suggested in the literature [34, 35 ]. This may also be the case in Hennoch-Schönlein´s purpura, a disease related to IgA nephropathy [36]. The epidemic described suggests either that the outbreak of four cases of glomerulonephritis was due to S. pyogenes but coincided with the transmission and colonization of S. constellatus or that S. constellatus strains were highly pathogenic or nephritogenic and that this organism can be effec- tively transmitted. Further studies on the possible role of S. constellatus in the pathogenesis of post-streptococcal glomerulonephritis [37] are warranted. ACKNOWLEDGEMENTS M Norgren Umeå, Sweden is acknowledged for the analysis of pulse field gel electrophoresis and PCR analysis of the ska gene as well as further typing of group C streptococci. I. Messel Oslo, Norway is thanked for the checkerboard analysis. The county council of Östergötland, Sweden is acknowledged for financial support. 229 REFERENCES 1. Villareal H Jr, Rico E, Zabriskie JB (1979) Streptococcus-related acute glomerulonephritis. Arch Inst Cardiol Mex 49: 89-102. 2. Van Buynder PG, Gaggin JA, Martin D, Pugsley D, Mathews JD (1992) Streptococcal infection and renal disease markers in Australian arboriginal children. Med J Aust 156: 537-540. 3. Glassock RJ, Cohen AH, Adler SC (1996) Primary glomerular diseases. In: Brenner BM, Rector FC(eds), The Kidney. WB Saunders company, Philadelphia 1394-1402. 4. Earle DP, Potter EV, Poon-King T, Finklea JF, Sharrett AR, Ortiz J (1970) Streptococcal skin infections and epidemic acute nephritis in Trinidad. Trans Am Clin Climatol Assoc 81: 184-195. 5. Barnham M, Thornton TJ, Lange K (1983) Nephritis caused by Streptococcus zooepidemicus (Lancefield group C). Lancet Apr 30; 1 (8331): 945-948. 6. Duca E, Teodorovici G, Radu C et al. (1969) A new nephritogenic streptococcus. J Hyg 67: 691- 698. 7. Reid HFM, Bassett DCJ, Poon-King T, Zabrieskie JB, Read SE (1985) Groop G streptococci in healthy school-children and in patients with glomerulonephritis in Trinidad. J Hyg Camb 94: 61-68. 8. Roth S, Andrassy K, Schmidt KH, Gunther F, Ritz E (1999) Febrile lady with acute renal failure and desquamating erythema. Am J Kidney Dis 34: 150-154. 9. Fujiyoshi T, Okasaka T, Yoshida M, Maishima K (2001) Clinical and bacteriological significan- ce of Streptococcus Milleri group in deep neck abscesses. Nippon Jibiinkoka Gakkai Kaiho; 104 (2): 147-156 (Japanese, Eng. Abstr.). 10. Fujiyoshi T, Inaba T, Udaka T, Tanabe T, Yoshida M, Makishima K (2001) [ Clinical significan- ce of the Streptococcus milleri group in peritonsillar abscesses] [Japanese, Eng. abstract]. Nippon Jibiinkoka Gakkai Kaiho 104: 866-871. 11. Whiley RA, Hall LM, Hardie JM, Beighton D (1999) A study of small-colony, beta-haemolytic, Lancefield group C streptococci within the anginosus group: description of Streptococcus con- stellatus subsp. pharyngis subsp. nov., associated with the human throat and pharyngitis. Int J Syst Bacteriol 49: 1443-1449. 12. Granlund M, Öberg L, Sellin M, Norgren M (1998) Identification of a novel insertion element, IS1548, in group B streptococci, predominantly in strains causing endocarditis. J Infect Dis 177: 967-976. 13. Johnston KH, Chaiban JE, Wheeler JC (1992) Analysis of the variable domain of the streptokina- se gene from streptococci associated with post streptococcal glomerulonephritis. In: Orefieci G, ed. New perspektives on streptococci and streptococcal infections. Stuttgart: Gustav Fischer Ver- lag: 339-341. 14. Extraction & purification protocols (1997) Blood & cultured cells. Animal tissues & paraffin sec- tions. Amersham Life Science: 18-19. 15. Socransky SS, Smith C, Martin L, Paster BJ, Dewhirst FE, Levin AE (1994) “Checkerboard” DNA-DNA hybridization. Biotechniques: 17: 788-792. 16. Westrhenen R v, J J Weening, Krediet RT (1998) Pneumonia and glomerulonephritis caused by mycoplasma pneumoniae. Nephrol Dial Transplant 13: 3208-3211. 17. Meyrier A (2001). Postinfectious glomerulonephritis. In: Primer on kidney diseases, 3d ed. Ed. Greenberg A. Academic press. San Diego and London 190-194. 18. Kaufhold A, Ferreri P (1993) The microbiologic aspects, including diagnosis, of beta-hemolytic streptococcal and enterococcal infections. Infect Dis North Am 7: 235-256. 19. Eriksson B, Jorup-Ronstrom C, Karkkonen K, Sjoblom AC, Holm SE (1996) Erysipelas: clinical and bacteriologic spectrum and serological aspects. Clin Inf Dis 23: 1091-1098. 20. Jansen TL, Janssen M, Traksel R, de Jong AJ (1999) A clinical and serological comparison of group A versus non-group A streptococcal reactive arthritis and throat culture negative cases of post-streptococcal reactive arthritis. Ann Rheum Dis 58: 410-414. 21. Cronin WJ, Lange K (1990) Immunologic evidence for the in situ deposition of a cytoplasmic streptococcal antigen (endostreptosin) on the glomerular basement membrane in rats. Clin Nep- hrol 34: 143-146. 230 22. Peake PW, Pussell BA, Karpius TE, Riley EH, Charlesworth JA (1991) Post-streptococcal glo- merulonephritis: studies on the interaction between nephritis strain-associated protein (NSAP), complement and the glomerulus. APMIS May; 99(5): 460-466. 23. Poon-King R, Bannan J, Viteri A, Cu G, Zabriskie JB (1993). Identification of an extracellular plasmin binding protein from nephritogenic streptococci J Exp Med 178: 759-763. 24. Nordstrand A, Norgren M, Holm SE (1999) Pathogenic mechanism of acute post-streptococcal glomerulonephritis. Scand J Infect Dis 31(6): 523-537. 25. Berge A, Kihlberg BM, Sjoholm AG, Bjorck L (1997) Streptococcal protein H forms soluble complement-activating complexes with IgG, but inhibits complement activation by IgG-coated targets. J Biol Chem 272: 20774-781. 26. Claridge JE 3rd, Attori S, Musher DM, Hebert J, Dunbar S (2001) Streptococcus intermedius, streptococcus constellatus and streptococcus anginosus (“Streptococcus milleri group”) are of different clinical importance and are not equally associated with abscess. Clin Infect Dis 32: 1511-1515. 27. Sunde PT, Tronstad L, Eribe ER, Lind PO, Olsen I (2000). Assessment of periradicular microbi- ota by DNA-DNA hybridisation. Endod Dent Traumatol 16: 191-196. 28. Jerng JS, Hsuch PR, Teng LJ, Lee LN, Yang PC, Luh KT (1997). Empyema thoracis and lung abscess caused by viridans streptococci. Am J Respir Crit Care Med 156: 1508-1514. 29. Baran J Jr, Abdo WM, Merrit KW, Khatib R (1998) Tricuspid valve endocarditis due to a mode- rately susceptible Streptococcus constellatus; persistent bacteremia and fatal outcome despite penicillin plus gentamicin therapy. Scand J Infect Dis 30: 420-421. 30. Arnold SB, Valone IA, Askenase PW, Kashgarian M, Freedman LR(1975). Diffuse glomerulo- nephritis in rabbits with Streptococcus viridans endocarditis Lab Invest 32: 681-689. 31. Neugarten J, Gallo GR, Baldwin DS (1984). Glomerulonephritis in bacterial endocarditis. Am J Kidney Dis 3: 371-379. 32. Neugarten J, Baldwin DS (1984). Glomerulonephritis in bacterial endocarditis. Am J Med; 77: 297-304. 33. Wanahita A, Goldsmith EA, Musher DM, Clarridge JE3d, Rubio J, Krishnan B, Trial J (2002). Interaction between human polymorphonuclear leucocytes and streptococcus milleri group bacte- ria. J Infect Dis; 185: 85-90. 34. Sagel I, Treser G, Ty A et al. (1973). Occurrence and nature of glomerular lesions after group A streptococci infections in children. Ann Intern Med 79: 492-499. 35. Okada K, Saitoh S, Sakaguchi Z et al. (1996). IgA nephropathy presenting clinicopathologic fea- tures of a post-streptococcal glomerulonephritis. Eur J Pediatr 155: 327-330. 36. Masuda M, Nakanishi K, Yoshizawa N, Iijima K, Yoshikawa N (2003). Group A streptococcal antigen in the glomeruli of children with Henoch-Schonlein nephritis. Am J Kidney Dis 41: 366- 370. 37. Rincon J, Viera NT, Romero MJ, Mosquera JA (2003). Increased production of chemotactic cytokines and elevated proliferation and expression of intercellular adhesion molecules in rat mesangial cells treated with erythrogenic toxin type B; its precursor isolated from nephritogenic streptococci. Nephrol Dial Transplant. Jun; 18: 1072-1078. Corresponding author: G. Almroth, MD, PhD. Department of Nephrology, University Hospital of Linköping, S-581 85 Linköping, Sweden. Phone +4613222000, Fax +4613224514, e-mail: gabriel.almroth@lio.se 231