Localized Mycobacterium avium complex infection in a patient on HAART

CORRESPONDENCE The world's ®rst case of Serratia liquefaciens intravascular catheter-related suppurative thrombophlebitis and native valve endocarditis Serratia liquefaciens is an organism rarely encountered in clinical practice. It belongs to the genus Serratia and the family Enterobacteriaceae [1]. It is widely distributed in nature, including river water [2], mineral, spring and table water [3], domestic sewage [4], ¢sh, minced meat and pasteurized milk or cream [5]. It has been reported as a cause of mastitis in a dairy herd [6]. In humans, it has rarely been reported as a cause of nosocomial infections, including urinary tract infection [7], pneumonia [8], post-sternotomy mediastinitis [9], neonatal meningitis [10] and septicaemia resulting from transfusion of contaminated blood products [11]. A few reports of S. liquefaciens causing community-acquired infections include peritonitis associated with continuous ambulatory peritoneal dialysis [12], pustulosis and costochondritis in heroin addicts [13], ¢stulous pyoderma [14], septic arthritis [15] and ocular infections resulting from contamination of contact lens cases [16]. I recently encountered a patient who developed an intravascular central catheter-related suppurative thrombophlebitis and endocarditis due to S. liquefaciens. He was a 55-year-old man who had developed short gut syndrome as a complication of chronic pancreatitis and multiple bowel resections. He had been dependent on parenteral nutrition for the preceding 6 months. He presented with fever, malaise and pain in the right side of the neck, along the tunnel of the indwelling intravenous central catheter. On physical examination, he had no heart murmur or peripheral signs of endocarditis. Four blood cultures grew S. liquefaciens within 48 h of inoculation. Duplex ultrasound revealed an occlusive clot in the right internal jugular vein surrounding the central venous catheter. Transesophageal echocardiography showed a mobile thrombus originating in the superior vena cava extending into the right atrium, measuring 5.2  0.9 cm, and all heart valves appeared normal. After a therapeutic anticoagulation was achieved, the central venous catheter was removed. He subsequently underwent resection of the right atrial mass, which on histological examination was con¢rmed to be a thrombus with secondary suppurative endocarditis. The organism was susceptible to multiple antibiotics, including piperacillin, trimethoprimsulfamethoxasole, third-generation cephalosporins, cipro£oxacin and imipenem-cilastatin. He was treated with ceftriaxone, 2 g daily for 4 weeks, for ease of administration with concomitant parenteral nutrition. To date, there have been no prior reports of suppurative thrombophlebitis or endocarditis caused by S. liquefaciens in the world's literature. This organism is di¡erentiated from S. marcescens by its relatively weak phospholipase activity [17] and the lack of fermentation of L -arabinose by the latter [1]. Anti-

microbial susceptibility is also somewhat di¡erent, with S. liquefaciens being more resistant to aztreonam, ceftazidime and amikacin than S. marcescens [18]. Endocarditis caused by S. marcescens is seen most commonly in intravenous drug users and in one report it caused 14% of all addict-associated endocarditis [19]. Among these patients, most cases of right-sided endocarditis were cured by antibiotics alone and most cases of left-sided endocarditis treated medically did not survive. Most other cases of endocarditis due to S. marcescens have occured in patients with prosthetic heart valves. Only two cases of native valve endocarditis due to S. marcescens were reported in the literature [20,21]. Both had right-sided involvement and indwelling intravenous catheters at the time of diagnosis and both were successfully treated medically. An experimental endocarditis rabbit model using S. marcescens con¢rmed the signi¢cance of the presence of an indwelling catheter in the development of endocarditis [22]. The classic presentation of suppurative thrombophlebitis involves the internal jugular vein in association with acute oropharyngeal infections ^ Lemierre's syndrome. However, several other veins can be a¡ected by the same problem, such as the pelvic veins with puerperal sepsis, the portal vein with in£ammatory bowel disease and the dural venous sinuses with contiguous infections. Suppurative thrombophlebitis of the central veins as a complication of central venous catheters is a common problem in patients receiving cancer chemotherapy or hyperalimentation. Unlike peripheral vein suppuration, surgical excision of central veins may not be feasible. Medical management with intravenous antibiotics, catheter removal and anticoagulation is a reasonable alternative, but in cases such as ours, surgical excision of a deep-seated focus of infection is recommended [23].

REFERENCES 1. Farmer JJ et al. Enterobacteriaceae. In: Balows A, Hausler JRWJ, Herrmann KL, Isenberg HD, Shadomy HJ, eds. Manual of Clinical Microbiology. Washington, DC: American Society for Microbiology, 1991; 360^83. 2. Merch-Sundermann V, Wundt W. Bacteriologic quality of water from the Rhine and its tributaries in the Rhine-Neckar region. I. Bacterial count and Enterobacteriaceae of the current status of pollution. Zentralblatt fur Bakteriologie und Hygiene ^ Serie B, Umwelthygiene, Krankenhaushygeine, Arbeitshygiene, Preventive Med 1987; 184: 459^69. 3. Schindler PR. Enterobacteria in mineral, spring and table water. Gesundheitswesen1994; 56: 690^3. 4. Sallal AK. Enumeration of pathogenic bacteria from sewage sludge in Kuwait. Microbios 1987; 52: 7^16. 5. Lindberg AM, Ljungh A, Ahrne S, Lofdahl S, Molin G. Enterobacteriaceae found in high numbers in ¢sh, minced meat and pasteurised milk or cream and the presence of toxin encoding genes. IntlJ Food Microbiol 1998; 39: 11^7.

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Clinical Microbiology and Infection, Volume 6 Number 10, October 2000

6. Bowman GL, HuestonWD, Boner GJ, Hurley JJ, Andreas JE. Serratia liquefaciens mastitis in a dairy herd. J Am Vet Med Assoc 1986; 189: 913^5. 7. Serruys-Schoutens E, Rost F, Depre G. A nosocomial epidemic of Serratia liquefaciens urinary tract infection after cystometry. EurJ Clin Microbiol 1984; 3: 316^7. 8. Matsui S, Nakazawa T. Clinical bacteriology and empiric therapy for hospital-acquired pneumonia in the elderly at a national leprosarium. JpnJ Thorac Dis 1994; 32: 851^5. 9. Katsumata T, Shimakura T, Nakano H, ShimamuraY, Ishitoya H. Curative report of post-sternotomy mediastinitis due to bacterofungal infection. J Jpn AssocThorac Surg 1994; 42: 95^100. 10. Fitzgerald P, Drew JH, Kruszelnicki I. Serratia: a problem in a neonatal nursery. Aust PaediatrJ 1984; 20: 205^7. 11. Boulton FE, Chapman ST,WalshTH. Fatal reaction to transfusion of red-cell concentrate contaminated with Serratia liquefaciens. Transfus Med 1998; 8: 1^3. 12. Rose TF, Ellis-Pegler R, Collins J, Small M. Oral pe£oxacin mesylate in the treatment of continuous ambulatory peritoneal dialysis associated peritonitis: an open non-comparative study. J Antimicrob Chemother 1990; 25: 853^9. 13. Altes J, Usandizaga I, Raya C, Forteza-Rei J. Pustulosis and costochondritis caused by Serratia liquefaciens in heroin addicts [letter]. Enfermedades InfecciosasY Microbiologia Clinica 1990; 8: 464. 14. Waldermann F, Maucher OM, Buck A, Goerttler E. Fistulous pyoderma caused by Serratia liquefaciens. Hautarzt 1987; 38: 36^9. 15. Harden WB, Fisher JF, Loebl DH, Bailey JP. Septic arthritis due to Serratia liquefaciens. Arthritis Rheum 1980; 23: 946^7. 16. Midelfart J, Midelfart A, Bevanger L. Microbial contamination of contact lens cases among medical students. CLAO J 1996; 22: 21^4. 17. Legakis NJ, Nicolas KJ, Xilinas M, Papavassiliou J. Di¡erentiation of Serratia marcescens and Serratia liquefaciens by tests for lipase and phospholipase production. J Med Microbiol 1978; 11: 225^31. 18. So¢anou D, Proya E, Kondodimou A. In-vitro susceptibility of clinical isolates of Serratia species to antimicrobial agents. J Chemother 1990; 2: 79^81. 19. Mills J, Drew D. Serratia marcescens endocarditis: a regional illness associated with intravenous drug abuse. Ann Intern Med 1976; 84: 29^35. 20. Hyams KC, Mader JT, Pollard RB, Parks DH, Thomson PD, Reinarz JA. Serratia endocarditis in a pediatric burn patient. Cure with cefotaxime. JAMA 1981; 246: 983^4. 21. Pearlman SA, Higgins S, Eppes S, Bhat AM, Klein JD. Infective endocarditis in the premature neonate. Clin Pediatr 1998; 37: 741^ 6. 22. Gutschik E, Norwood RS, MÖller S, Olling S. Experimental endocarditis in rabbits. Acta Pathol Microbiol Scand Section B Microbiol 1980; 88: 269^76. 23. Verghese A, Widrich WC, Arbeit RD. Central venous septic thrombophlebitis: the role of medical therapy. Medicine 1985; 64: 394^400.

S. B. Mossad Cleveland Clinic Foundation, 9500 Euclid Avenue, S-32, Cleveland Ohio 44195, USA Tel: ‡1-216 - 445-2572 Fax: ‡1-216 - 445-9446 E-mail: [email protected]

Pericarditis caused by Corynebacterium urealyticum Corynebacterium urealyticum, formerly known as Corynebacterium group D2 or CDC group D2 [1], is an aerobic, catalase-positive, Gram-positive bacillus which shows resistance to multiple antibiotics. It has been associated mainly with infections of the urinary tract [2], and the isolation of this organism in other infections is very unusual.We report the ¢rst case, to our knowledge, in which C. urealyticum is associated with pericarditis. A 55-year-old woman with a medical history of diabetes mellitus was admitted to the emergency room with a 2-week history of lower retrosternal pain and dyspnea. On examination she was pyrexial (38  C) and tachypneic. There were no pericardial or pleural rubs.The patient was alert and orientated. Results of laboratory studies were as follows: hemoglobin level, 10.4 g/dL; white blood cell count, 9910/mm3 (79% neutrophils, 13% lymphocytes, 6.9% monocytes and 0.9% eosinophils); erythrocyte sedimentation rate, 56 mm/h. The urea and electrolyte values were normal. The plasma glucose was elevated at 296 mg/dL. The electrocardiogram (ECG) and chest X-ray were normal. Two-dimensional echocardiograms revealed mild cardiomegaly with normal myocardial contractibility, and a big pericardial e¡usion with ¢brinous bands; no echocardiographic signs of cardiac tamponade were observed. A diagnosis of acute pericarditis was made, and pericardiocentesis was prescribed. Seven hundred milliliters of a purulent pericardial £uid were obtained. Histologic examination and Gram stain of the £uid only showed many polymorphonuclear cells. Cultures were performed on blood and chocolate agar, McConkey agar, Sabouraud agar and Brucella agar. An aerobic, catalase-positive bacillus with the typical appearance of diphtheroids grew in pure culture in blood and chocolate agar. The organism was initially identi¢ed as C. urealyticum by the API Coryne System (BioMerieux, Mazcy l'Etoile, France). Conventional methods of identi¢cation con¢rmed that the organism was C. urealyticum [1]. The isolate was characterized by the strong urease activity and the inability to ferment glucose or reduce nitrate. Antimicrobial susceptibility tests were performed by the Kirby ^ Bauer disk di¡usion method on Mueller ^ Hinton agar with 5% sheep blood. The organism was susceptible to rifampicin, teicoplanin and vancomycin, and resistant to penicillin, imipenem, gentamicin, erythromycin and cipro£oxacin. After treatment with intravenous vancomycin for 2 weeks, the patient made a rapid and full recovery. An echocardiogram on day 15 of admission was normal, with no evidence of pericardial e¡usion. The blood cultures as well as the urine cultures were repeatedly negative. Investigation for mycobacteria, fungi and viruses was negative. C. urealyticum has been reported to frequently colonize the skin of hospitalized patients [3]. Despite frequent coloniza-

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Correspondence

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tion, C. urealyticum rarely causes infection outside of the urinary tract. A limited number of non-urinary infections caused by this organism have been reported: some cases of endocarditis [4^6], bacteremia [7^11], postsurgical osteomyelitis [12], peritonitis in outpatients with chronic peritoneal dialysis [13], necrotic infection of soft tissue [14] and wound infections [8]. Most of these infections were hospital-acquired, the patients had been previously treated with antibiotics, and the infection, in many cases, was related to previous surgery or invasive manipulations. To our knowledge, this is the ¢rst case of a pericarditis caused by C. urealyticum. In a literature search for the period 1984^99 via MEDLINE, no other case was found. Our case involved pericarditis in a diabetic patient who had not been previously treated with antimicrobial agents and had not acquired the infection in the hospital, and on whom no invasive diagnostic procedure or surgery had been previously performed. Due to the unusual localization of this infection, establishing the pathogenicity of this microorganism may be di¤cult. C. urealyticum was the only organism isolated from the pericardial £uid, and since the symptoms resolved after the organism was eliminated by antibiotic treatment, we believe that this bacterium was the etiologic agent of the infection. Diabetes was the only underlying predisposing factor that may contribute to the pathogenicity of this strain.

10. Aracil B, Perea B, Barros C, Gomez-Garces JL. Bacteriemia causada por Corynebacterium urealyticum en 2 pacientes coinfectados con HIVy HBV. Enferm Infecc Microbiol Clin 1997; 15: 172^3. 11. Wood CA, Pepe R. Bacteriemia in a patient with non-urinarytract infection due to Corynebacterium urealyticum. Clin Infect Dis 1994; 19: 367^8. 12. Chomarat M, Breton P, Dubost J. Osteomyelitis due to Corynebacterium group D2. EurJ Clin Microbiol Infect Dis 1991; 10: 43. 13. Van Bosterhaut B, Claeys G, Gigi J,Wauters G. Isolation of Corynebacterium group D2 from clinical specimens. Eur J Clin Microbiol Infect Dis 1987; 6: 418^19. 14. Saavedra J, Rodriguez JN, Fernandez-Jurado MD et al. A necrotic soft-tissue lesion due to Corynebacterium urealyticum in a neutropenic child. Clin Infect Dis1996; 22: 851^2.

REFERENCES

Endocarditis caused by anaerobic and micro-aerophilic bacteria accounts for 7^10% of all cases of infectious endocarditis [1]. The most frequently reported causes of anaerobic endocarditis are Bacteroides (particularly the B. fragilis group), and anaerobic Streptococcus, Clostridium, Peptostreptococcus, Fusobacterium, Propionibacterium and Lactobacillus species occasionally cause endocarditis [2]. Only 25 cases of clostridial endocarditis have been reported to date, and the most common species was C. perfringens [3^7]. To our knowledge, this case is the ¢rst occurrence of Clostridium histolyticum endocarditis reported in the literature. An 18 -year-old woman was admitted to the Turgut Ozal Medical Center (Malatya,Turkey) following a 20 -day history of cough, yellowish-green sputum, and fever; however, she had not experienced dyspnea. She denied smoking and use of drugs. On physical examination, the patient appeared toxic, with an axillary temperature of 39  C; pulse rate of 85/min, blood pressure of 90/60 mmHg, and respiratory rate of 20/ min. Awidespread systolodiastolic murmur could be heard on chest auscultation. Auscultation of the lungs revealed crepitant rales over the basal areas, but no pleural rub was noticed. No other anomalies were detected, and pelvic examination did not reveal any foreign body.The results of a neurologic exami-

1. Pitcher D, Soto A, Soriano F, Valero-Guille¨ n P. Classi¢cation of coryneform bacteria associated with human urinary tract infection (group D2) as Corynebacterium urealyticum sp. nov. IntJ Syst Bacteriol 1992; 42: 178^81. 2. Soriano F, Aguado JM, Ponte C, Fernandez-Roblas R, Rodriguez-Tudela JL. Urinary tract infection caused by Corynebacterium Group D2: report of 82 cases and review. Rev Infect Dis 1990; 12: 1019^34. 3. Soriano F, Rodriguez-Tudela JL, Fernandez-Roblas R, Aguado JM, Santamaria M. Skin colonization by Corynebacterium groups D2 and JK in hospitalized patients. J Clin Microbiol 1988; 26: 1878^ 80. 4. Langs JC, De Briel D, Sauvage C, Blickle JF, Akel H. Endocardite a© Corynebacterium du group D2, a© point de de¨ part urinaire. Med Mal Infect 1988; 5: 293^5. 5. Ena J, Berenguer J, Pelaez T, Bouza E. Endocarditis caused by Corynebacterium group D2. J Infect 1991; 22: 95^6. 6. Notario R, Borda N, Gambade T. Endocarditis en valvula protesica causada por Corynebacterium urealyticum. Med (B Aires) 1996; 56: 57^8. 7. Marshall RJ, Routh KR, MacGowan AP. Corynebacterium CDC group D2 bacteraemia. J Clin Pathol 1987; 40: 813^14. 8. Soriano F, Ponte C, Ruiz P, Zapardiel J. Non-urinary tract infections caused by multiply antibiotic-resistant Corynebacterium urealyticum. Clin Infect Dis 1993; 17: 890^1. 9. Buendia B, Delgado T, Lo¨pez S, Vallejo P, del Rey MC, LopezBrea M. Bacteriemia transitoria por Corynebacterium urealyticum: a propo¨sito de 2 casos. Enferm Infecc Microbiol Clin1996; 14: 367^9.

M. Ojeda-Vargas, M. A. Gonzalez-Fernandez, D. Romero, A. Cedre¨ s and C. Monzo¨ n-Moreno* *U.D. Microbiolog|¨ a, Centro de Ciencias de la Salud, ULPGC, Gran Canaria, Islas Canarias, Spain Tel: ‡ 34 928 45 34 05 Fax: ‡ 34 928 45 14 13 E-mail: [email protected]

Infective endocarditis due to Clostridium histolyticum

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Clinical Microbiology and Infection, Volume 6 Number 10, October 2000

nation were normal. Laboratory data included a WBC count of 11 10 9/L, a hemoglobin level of 8.7 g/dL, and an erythrocyte sedimentation rate of 80 mm/h. The results of the platelet count, prothrombin time, partial thromboplastin time, bleeding time, blood urea nitrogen and serum creatinine, glucose, biluribin, calcium, protein, albumin, globulin, electrolytes, aspartate, aminotransferase, alanine transaminase and alkaline phosphatase and urine sediment were within normal ranges. Lactate dehydrogenase was 723 IU/L. Serologic tests for HIV, HBV and HCV performed upon admission were all negative. A chest X-ray and abdominal ultrasonography did not reveal any abnormality. The electrocardiogram showed changes due to left ventricular hypertrophy. An echocardiogram showed aortic regurgitation, prominent thickening and a large vegetation on the aortic valve, a ¢nding suggestive of infective endocarditis. Two subsequent aerobic blood cultures, sputum cultures and urine cultures were all negative. Anaerobic blood culture was not performed. The patient received treatment with penicillin 4 million units every 4 h plus 80 mg of gentamicin every 8 h empirically. Five days after admission, cardiac surgery was planned due to congestive cardiac failure and large mobile vegetations on the aortic valve. Surgery also revealed a large vegetation on the aortic valve. A microbiological specimen from the valve tissue sent to the laboratory for culture was inoculated on Brucella blood agar plates for anaerobic culture and tryptic soy ^ blood agar plates for aerobic culture. Gram-positive rods were seen on Gram staining of the direct preparation. Growth occurred after 48 h under anaerobic conditions. The aerotolerance test, cellular morphology, susceptibility to kanamycin, colistin and vancomycin special potency disks for anaerobic bacteria and spore tests revealed a Clostridium sp. [8]. Species identi¢cation and antibiotic susceptibility tests were performed in the Anaerobe Reference Laboratory in Helsinki. The organism was identi¢ed as C. histolyticum, and its susceptibilities to penicillin, metronidazole, clindamycin and imipenem were determined by means of the E test (AB Biodisk, Solna, Sweden), and the following MICs were obtained: penicillin G, 0.016 mg/L; metronidazole, 0.094 mg/L; clindamycin, 0.016 mg/L; and imipenem, 0.012 mg/L. These MICs are below the breakpoint values [4]. Treatment with penicillin G plus metronidazole was started in the immediate postoperative period. After a 5-week treatment period, laboratory and clinical ¢ndings were all normal. C. histolyticum is known as one of the histotoxic clostridia, and is most commonly involved in myonecrosis [9]. This species of Clostridium has not been previously isolated from endocarditis or bacteremia patients. There is a propensity for anaerobic endocarditis to produce large vegetations. Diagnosis of anaerobic endocarditis is not di¡erent from that of other forms of endocarditis and usually involves demonstration of continuous bacteremia and a valvu-

lar lesion that is consistent with endocarditis [2]. In our case, the echocardiogram showed a large vegetation on the aortic valve which was also reported by the surgeon. In the histologic examination, valvular endocardial tissue showed mild plasma cell in¢ltration with scattered eosinophils. Small foci of ¢brinous vegetations were also present on the valvular surface. All of these ¢ndings were consistent with valvular endocarditis which was also previously hyalinized. Of the 25 cases reported in the literature, 14 have detailed clinical information for the major characteristics of clostridial endocarditis. Two were associated with intravenous drug abuse, and subacute presentation was documented in six. Carcinoma of the colon or cervix was documented in two other cases. Peripheral emboli were reported in four cases, coexistent atypical pneumoniae and cardiac failure was documented in one case [5], emboli were documented in one case [6], and hemiplegia was reported in one case [4]. An echocardiogram indicated vegetations on the tricuspid valve [3], on the aortic wall at the junction with a dacron prosthesis [4], and below the mitral annulus of the left ventricular wall [6]; also, in the other study, an echocardiogram showed a para-aortic abscess [7]. Vegetations were revealed at autopsy on the tricuspid and pulmonary valves in another case [5]. Involvement of the tricuspid valve, the mitral valve, the aortic valve or both was documented in all other cases; infection of prosthetic valves was reported in four cases [4,5,7]. Negative aerobic blood cultures are sometimes associated with fungal or anaerobic endocarditis [9]. Con¢rming our results, the aerobic blood cultures were all negative. Using both aerobic and anaerobic blood culture bottles together will successfully increase the rate of detection of anaerobic etiologic agents of bacteremia or infective endocarditis. Although anaerobic blood culture was not performed, isolation of C. histolyticum from valvular tissue specimens and histology of the surgically resected valvular tissue indicated that this bacterium was the etiologic agent of this patient's infective endocarditis, con¢rming the clinical ¢ndings. ACKNOWLEDGMENTS We thank Dr Hannale Somer-Jousimies and her colleagues in the Anaerobe Reference Laboratory in Helsinki for identi¢cation to species level and antibiotic susceptibility testing of Clostridium histolyticum.

REFERENCES 1. Scheld WM, Sande MA. Endocarditis and intravascular infections. In: Mandel GL, Douglas RG, Bennett JE, eds. Principles and practice of infectious diseases. NewYork: Churchill Livingstone, 1995: 740^3.

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Correspondence

2. Finegold SM, GeorgeWL, Mulligon ME. Anaerobic infections. Chicago:Year book Medical Publishers, 1986. 3. Moyano R, Gomez-Mateos M, Lozanode Leon F, Florez C, Jimenez-Ocana C, Gamboa F. Clostridium bifermentas: an exceptional agent of endocarditis. Clin Infect Dis1994; 18: 837. 4. Mendes GM, Oplistil CP, Santos TJL, Mandy C. Clostridium perfringens as a cause of infectious endocarditis in a patient with a vascular prosthesis. Clin Infect Dis1996; 22: 866^7. 5. Cutrona AF,Watanakunakorn C, Schaub CR, Jagetia A. Clostridium innocuum endocarditis. Clin Infect Dis 1995; 21: 1306^7. 6. Holland II F, Fernandez L, Jacops J, Blooki H. Clostridial endocarditis following penetrating cardiac trauma. Clin Infect Dis 1997; 24: 87^8. 7. Robles P, Gorcia-Gallego F, de Alba J, Gorcia J, Dominguez FJ, Oliver JM. Prosthetic endocarditis and splenic abscess caused by Clostridium clostridioformis. Rev Esp Cardiol 1997; 50: 360^2. 8. Summanen P, Baron EJ, Citron DM, Strong CA, Wexler HM, Finegold SM.Wadsworth anaerobic bacteriology manual. Belmont: Star Publishing Company, 1993. 9. Oksana M, Korzeniowski DK. Endocarditis. In: Gorbach SL, Barlett JG, Blacklow NR, eds. Infectious diseases. Philadelphia: WB Saunders Company, 1998: 663^74.

B. Durmaz*, H. E. Agel, E. So«nmez, R.Tu«rko«z and E. Aydin *Department of Microbiology, Faculty of Medicine, Ino«nu« University, 44069 Malatya,Turkey Tel: ‡ 422 341 0182/0660 Fax: ‡ 422 341 0728 E-mail: [email protected]

Susceptibility pattern of Streptococcus pneumoniae in outpatients in Germany During a 1992^97 German surveillance study, Reinert et al [1] found no penicillin-resistant Streptococcus pneumonaie strains

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among blood culture isolates (n ˆ 200).We report the susceptibility pattern of recent S. pneumoniae strains (only one per patient) isolated from outpatients seen in general practice or in our Outpatient Department.The majority of the S. pneumoniae strains (n ˆ 89) were cultured from material sent to a laboratory serving general practitioners in the Frankfurt area (EJKR) in 1998. Only eight strains (three of them in winter 1999) were cultured from patients who consulted the Outpatient Department of our Hospital. The sources of the strains were: upper respiratory tract (63); lower respiratory tract (17); blood/cerebrospinal £uid (6); ear (5); others (6). Minimum inhibitory concentrations (MIC) were determined for penicillin (PEN), erythromycin (ERY), doxycycline (DOXY), clindamycin (CLIN), levo£oxacin (LEV) and moxi£oxacin (MOX) using the E test method. Three S. pneumoniae strains, with a penicillin MIC of 0.5, 2.0 and 4.0 mg/L, respectively, provided by Dr Bryskier, France were used as quality control strains. The MIC distribution of the 97 strains tested is given in Table 1. The rate of resistance at break-points de¢ned in the Alexander Study [2] to PEN is 4.1%, to ERY is 15.5%, and to DOXY is 15.5%. As recommendations from an independent source such as DIN (Deutsches Institut fuer Normung e.V.) are still missing, we used the break-points suggested by several German pharmaceutical companies: MOX, R1mg/L for sensitivity, r4 mg/L for resistance; LEV, R 2 mg/L for sensitivity, r8 mg/L for resistance. At these admittedly high breakpoints for the newer quinolones none of the strains was classi¢ed as resistant (Table 2). Four out of 97 strains which we collected in 1998^99 were resistant to penicillin.We believe this is the highest incidence of penicillin-resistant pneumococci reported so far from Germany. In the present study we found 15.5% (15/97) erythro-

Table 1 Number of strains inhibited at given concentrations (E test dilutions in mg/L) Drug Name PEN ERY DOXY CLIN MOX LEV Drug Name PEN ERY DOXY CLIN MOX LEV

0.006

0.008

0.012

0.016

0.023

0.032

0.047

1

1

43

27

10

1 1

2

1.5 2 1 1

2 2

3

4 1 1

1

1

6

1

8

3

12

0.064

0.094

0.125

0.19

10 2 10

2 13 2 23 1

3 43 9 28 17

14 7 13 39

16

24

32

64

4

3

3

2

0.25

0.38

3

1

8 2 37 1 128

16

r256

13 36

7

1

PEN, Penicillin G; ERY, erythromycin, DOXY, doxycyclin; CLIN, clindamycin; MOX, moxi¯oxacin; LEV, levo¯oxacin.

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0.75

1 1

30 1

4

1

7

16

29

3

14 3

0.5

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Clinical Microbiology and Infection, Volume 6 Number 10, October 2000

Table 2 Breakpoints, R I S distribution, geom. mean and range of 97 pneumococcal strains Drug name

Breakpoints

No. of strains

%R

%I

%S

MIC50

MIC90

Geom Mean

Range

PEN ERY DOXY CLIN MOX LEV

Sˆ0.006; Rˆ2 Sˆ0.0.5; Rˆ4 Sˆ0.2; Rˆ8 Sˆ0.0.5; Rˆ4 Sˆ0.1; Rˆ4 Sˆ0.2; Rˆ8

97 97 97 97 97 97

4.1 14.4 15.5 13.4 0 0

9.3 1.0 1.0 0 0 8

86.6 84.5 83.5 86.6 100 92

0.023 0.125 0.5 0.125 0.19 1

0.125 512 16 512 0.25 1.5

0.03 0.38 0.64 0.32 0.20 1.12

0.00±2 0.047±512 0.064±64 0.016±512 0.094±0.38 0.25±3

PEN, Penicillin G; ERY, erythromycin; DOXY, doxycyclin; CLIN, clindamycin; MOX, moxi¯oxacin; LEV, levo¯oxacin.

mycin resistance, which is markedly higher than the 3% (only six out of 200 strains collected between 1992 and 1996) reported recently by Reinert et al [3] for Germany, and re£ects the trend reported by Kresken et al [4] who observed an increase in erythromycin resistance (break-point r1mg/L, which is lower than the one used in the Alexander Project [2]) from 3.6% in 1992 to 11.3% in 1997. Frankfurt (if not Germany) seems to have joined the `club of countries with penicillin-resistant pneumococci'. S.J. Korn, S. M. Rau¢,E. J. K. Rosenthal and P. M. Shah* *Klinikum der J.W. Goethe Universita« t, Zentrum der Inneren Medizin, Schwerpunkt Infektiologie, D- 60590 Frankfurt, Germany Tel: ‡49 69 6301 6614 Fax: ‡49 69 63017707 E-mail: [email protected] These results were presented at 21st ICC, Birmingham, 1999. Poster NO:190.

REFERENCES 1. Reinert RR, Schlaeger JJ, Lu«tticken R. Moxi£oxacin: a comparison with other antimicrobial agents of in-vitro activity against Streptococcus pneumoniae. JAntimicrob Chemother 1998; 42: 803^6. 2. Felmingham D, Gru« neberg RN. A multicentre collaborative study of the antimicrobial susceptibility of community-acquired, lower respiratory tract pathogens: The Alexander Project. J Antimicrob Chemother 1996; 38(Suppl.A): 1^57. 3. Reinert RR, Huber K, Lu«tticken R. Grepa£oxacin: comparison of di¡erent methods for MIC determination of Streptococcus pneumoniae [poster no. 881]. In: 9th ECCMID, Berlin, Germany, 1999. 4. Kresken M, Hafner D,Witte W, Reinert R. Resistenzentwicklung bei Staphylokokken und anderen grampositiven Erregern gegenu«ber Chemotherapeutika im mitteleuropa« ischen Raum. ChemotherapieJournal1999; 8(4): 136^44.

Localized Mycobacterium avium complex infection in a patient on HAART The increases in CD4 T-lymphocytes count induced by antiretroviral therapy provide an anti-infective protection such that primary prophylaxis against opportunistic infections can be stopped. There is, however, concern that CD4 count augmented by drug therapy may not include cells speci¢cally active against some of the frequent opportunistic infections, including atypical mycobacterial infections [1]. We report a case of Mycobacterium avium intracellulare occuring in a 55-year-old retired bank manager who had been unwell for about 9 months with repeated respiratory chest infections and loss of weight. He was thought initially to have an underlying neoplasm and had undergone extensive investigations including a whole body CT (computed axial tomographic) scan at the referring hospital. He was divorced in 1978 and during the 1980s had multiple homosexual contacts. During the 2 weeks prior to being referred he had become progressively short of breath associated with a dry cough. Physical examination was essentially normal apart from a temperature of 37.5  C. His chest X-ray revealed bulky hilar lymphadenopathy and there were patchy opacities of both the mid-zones. Blood gas analysis revealed pO2 10.6 kPa (4.5^6.1) and pCO2 4.1kPa (12.0^14.0). Pulmonary function tests showed that the TLCO was 6.77 mmol/min/kPa (62% of expected) and kCO was 1.18 mmol/min/kPa/L (84% of expected). Although induced sputums taken for indirect immuno£uorescent antibody stain against Pneumocystis carini were negative, a subsequent broncho-alveolar lavage examination was strongly positive. Acid-fast bacilli were not demonstrated. He was commenced on high dose cotrimoxazole (120 mg/kg per day) with prednisolone. He was counselled and consented to be tested for HIV and this was shown to be positive (ELISA Abbot (Abbot Laboratories, Chicago, USA); ELISA Organon (Organon Teknika Ltd, Boxtel, the Netherlands) and INNOLIA Blot test (Ghent, Belgium)). The HIV load was noted to

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Figure 1 CT scan demonstrating the localized cavitation of the lung due to Mycobacterium avium complex.

be 750 000 copies/mL (Roche Amplicor) with the CD4 count of 63 cells/uL (600^1100). He symptomatically improved with treatment but became very confused and disorientd on completion of the therapy for PCP, and a CT scan of the brain revealed a generalized cerebral atrophy. A lumbar puncture showed the protein elevated at 1.44 g/L (0.18^0.5) but there were no cells or organisms seen and the cryptococcal antigen (Latex agglutination test) was negative. He was commenced on anti-retroviral therapy of zidovudine, lamividine and indinavir. He was discharged home but had an episode of mania requiring treatment with haloperidol and lorezapam with good response. He clinically improved but 2 months later he developed a dry, unproductive cough with sweats and a fever. He was noted to be anemic with the hemoglobin of 9.9 (13.5^18.0 g/ dL). A repeat chest X-ray showed a nodular opacity at the apex of the right lower lobe. A CT scan of the thorax (Figure 1) demonstrated mediastinal lymphadenopathy and a patchy area of con£uent nodular opaci¢cation with a cavity in the apical segment of the right lower lobe. Broncho-alveolar £uid examination showed presence of acid-fast bacilli by Ziel^ Neilsen stain, and a trans-bronchial biopsy of the lesion showed granulomatous in£ammation with central necrosis containing polymorphs. Subsequent culture (MB\BacT OrganonTeknika Ltd) of the lavage £uid grew M. avium complex.Three sets of blood cultures did not show any growth. At this stage the HIV viral load was reduced signi¢cantly to 1850 copies/mL with a CD4 count of 250 cells/uL. The polymerase chain reaction (PCR) for M. tuberculosis was negative. The

patient was commenced on treatment with azithromycin and rifabutin. The organism was multiresistant to isoniazid, ethambutol, rifampicin, pyrazinamide, cipro£oxacin, azithromycin and streptomycin (resistance ratio method). His symptoms resolved and he was discharged home. Our case demonstrates the ever increasing spectrum of clinical manifestations associated with opportunistic infections in HIV-infected individuals in the era of HAART (Highly Active AntiRetroviral Therapy). Initiation of combination anti-retroviral therapy results in the dramatic decrease of the HIV viral load with a concomitant restoration of cellmediated immune function [1]. This enhanced immunity may lead to a marked in£ammatory reaction and to altered clinical manifestations of pre-existing latent infections. A recent study reported the incidence of pathogen-associated in£ammatory diseases occuring in 143 HIV-infected patients after treatment with potent anti-retroviral therapy. Thirty-three responders (25%) had one or more episodes of opportunistic infections. Five had localized MAC disease, six had exacerbation of CMV retinitis, nine had mucocutaneous herpes, eight had dermatomal zoster, four had myelitis and/or encephalitis with presumptive HSV infection, three had HCV-associated hepatitis, two had MTB-associated lymphadenitis/pneumonitis or cerebritis and six had in£amed molluscum contagiosum or warts. These diseases occured during the ¢rst 2 months of therapy and a low baseline CD4 T-cell count was the only predictor of their onset [2]. There have been a number of reports of a clinical syndrome of MAC-related in£ammatory lymphadenitis on initiating

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Clinical Microbiology and Infection, Volume 6 Number 10, October 2000

HAART. Race et al recognized this on initiation of a protease inhibitor, indinavir, in patients with advanced HIV-1 disease [3]. A retrospective study conducted on 52 patients with mycobacterial lymphadenitis in HIV showed 12 patients developed the infection within 12 weeks of initiating combination antiretroviral therapy. The clinical presentation, which may vary, was in these cases of localized disease and was associated with a relatively high CD4 count. All patients showed a good immunological and virological response to HAART [4]. A further analysis identi¢ed ¢ve patients who developed mycobacterial lymphadenitis within 8 weeks of initiating HAART. All patients had fever, weakness and splenomegaly. Four of the ¢ve patients were anemic with Hb < 10 g/dL and demonstrated lymphadenopathy. This was cervical in three, submandibular in two, mediastinal in three and preauricular in one [5]. Interestingly, our patient presented with a primary lung lesion (Figure 1).We are not aware of any other literature describing cavitatory lung lesions, due to localized MAC infection, relating to HAART therapy in HIV infection. MAC infections may re£ect restoration of pathogen-speci¢c immune responses rather than reactivation of the initiating infection. Partial immune reconstitution, as demonstrated by an increase in the CD4 counts on anti-retroviral therapy is likely to be responsible for the localized MAC infection. This reconstitution may lead to an unmasking of subclinical infection and an enhanced immune response to mycobacterial antigens. There is evidence to show that immune reconstitution due to HAARToccurs in two stages and the initial CD4 lymphocyte proliferation is primarly of the memory subsets [6^8]. Thus this marked memory T-cell-mediated and antigen-speci¢c in£ammatory reaction may then lead to tissue suppuration and granuloma formation, the hallmarks of mycobacterial disease. The occurence and severity of illness in our patient with localized but untreated MAC infection after the start of HAART suggests that patients with advanced HIV disease should be assessed for active mycobacterial infection before the initiation of such therapy. It is also important to consider that dual opportunistic infections may occur in advanced disease as our patient demonstrated. Therefore, patients with severe immune dysfunction should be monitored very carefully, in particular during the ¢rst weeks after initiating of HAART. Indeed, it may be important in the ¢rst few months of HAART to maintain patients on prophylaxis for opportunistic infections. H.Thaker and E. L. C. Ong Department of Infection& Tropical Medicine, University of Newcastle Medical School, Newcastle General Hospital, Newcastle uponTyne NE4 6BE UK E-mail: [email protected]

REFERENCES 1. Ong ELC. Prophylaxis against disseminated Mycobacterium avium complex in AIDS. J Infect 1999; 38: 6^8. 2. French M, Lenzo N, John M, Mallal S, Price P, Flexman J. Immune restoration disease after treatment of immunode¢cient HIVinfected patients with potent anti-retroviral therapy [Abstract 340/ 22323]. Int Conf AIDS, 1998; 12: 327^8. 3. Race EM, Adelson-Mitty J et al. Focal mycobacterial lymphadenitis following initiation of protease- inhibitor therpy in patients with advanced HIV-1 disease. Lancet 1998; 351: 252^5. 4. Phillips P, Kwiatkowski MB, Copland M, Craib K, Montaner J. Mycobacterial lymphadenitis associated with the initiation of combination antiretroviral therapy. JAcquir Immune De¢c Syndr Hum Retrovirol 1999; 20(2): 122^8. 5. Ho¡mann C, Horst HA, Degen O, Van Lunzen J, Stellbrink HJ. Manifestation of mycobacterial infection after initiating of highly active antiretroviral therapy (HAART) [Abstract 22172]. Int Conf AIDS 1998; 12: 296. 6. Pontesilli O, Kerkhof-Garde. S et al. Functional lymphocyte reconstitution and HIV-1 speci¢c T-cell responses during highly active antiretroviral therapy (HAART) [Abstract 31134]. Int Conf AIDS 1998; 12: 113. 7. Mezzaroma E, Pinter G, Cunsolo L et al. Lymphocyte subsets and functional changes in HIV-1 infected patient during HAART [Abstract 12302]. Int Conf AIDS 1998; 12. 8. Oliver Lantz F, Martinon I, Peguillet A et al. Longitudinal analysis of the immune reconstitution after HAART initiation in previously untreated patients [Abstract 21123]. Int Conf AIDS 1998; 12.

Endocarditis due to Gemella haemolysans in a patient with hemochromatosis Gemella haemolysans is a facultative anaerobic Gram-positive coccus that can be a commensal of the upper respiratory tract and oral cavity of humans. The clinical spectrum of infections produced by this microorganism includes endocarditis, septicemia and meningitis.We report a case of endocarditis due to G. haemolysans in a patients with hemochromatosis. A 77-year-old man with a long history of hemochromatosis with chronic liver disease and arterial hypertension was admitted to hospital because of malaise, anorexia, weight loss and progressive dyspnea over the previous 4 months. On admission he was afebrile and the physical examination revealed a grade 2 diastolic ejection murmur in the left sternal border and hepatomegaly. Laboratory studies disclosed the following data: leukocyte count 12 000/mm 3 (80% neutrophils, 17% lymphocytes), hematocrit 48%, erythrocyte sedimentation rate of 54 mm/h, iron 200 mg/dL, ferritin 2277 U

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Table 1 Summary of patients with reported cases of G. haemolysans endocarditis

Case/Reference

Age/Sex

Underlying conditions or source of infection

Infected valve

1/1

62/M

Dental manipulation

Mi

2/1

48/M

3/1 4/2 5/3

56/M 68/M 47/M

6/4

62/F

Poor dentition, respiratory viral syndrome Mi insuf®ciency, dental manipulation Ao insuf®ciency Prosthetic aortic valve, periodontal disease None

7/5

74/M

Mi prolapsed

Mi

8/6

42/M

Ao insuf®ciency, wound

Ao

9/7 10/8 11/9 12/10

71/M 53/F 20/M 34/M

Mi Mi Ao Ao

13/11 14/12

48/M 63/M

15/13

26/M

Present report

77/M

Colonic carcinoma Mi regurgitation None Prosthetic valve, endocarditis, dental sepsis Prosthetic Ao and Mi valves Chronic obstructive bronchitis, poor dentition Down syndrome, Ao insuf®ciency, ventricular septal defect Ao insuf®ciency, hemochromatosis

Treatment

Outcome Survived

Ao

Cefamandole, gentamicin, benzylpenicillin, amoxicillin Benzylpenicillin, gentamicin

Survived

Mi Ao Ao

Benzylpenicillin, gentamicin Benzylpenicillin, streptomycin Erythromycin, rifampin

Survived Survived Survived

Mi

Survived

Mi Mi

Benzylpenicillin, tobramycin, clindamycin Benzylpenicillin, gentamicin, amoxicillin Vancomycin, fusidic acid, amoxicillin, gentamicin Amoxicillin, clavulanic acid, gentamicin Benzylpenicillin, gentamicin Benzylpenicillin, gentamicin Cefuroxime, tobramycin, cipro¯oxacin, erythromycin Benzylpenicillin, gentamicin Amoxicillin, amikacin

Ao

Benzylpenicillin, gentamicin

Survived

Ao

Benzylpenicillin, tobramycin

Survived

Survived Survived Survived Survived Survived Survived Survived Survived

Note: Sex (M, Male; F, Female);Valves (Ao, Aortic valve; Mi, Mitral valve).

mg/L, AST 64 U/mL and ALT 57 U/mL. Radiographs of the chest were normal. Echocardiography revealed a vegetation on the aortic valve with moderate valvular insu¤ciency. G. haemolysans was isolated in the three blood cultures taken on admission. The identi¢cation was performed on the basis of Gram stain, catalase activity and biochemical characteristics (API 20 STREP, BioMe© rieux, France).The isolate was susceptible to penicillin (MIC: 0.06 mg/L). The patient was treated with penicillin G (24 MU IV daily, divided in six doses) and tobramycin (100 mg twice a day IV) for 2 weeks whereupon he showed improvement of his clinical status as well as sterilization of the blood cultures. In a follow-up visit 30 days after discharge, the echocardiogram did not show any vegetation. G. haemolysans was ¢rst described in 1938 by Thjo«tta and Bo«e [1] as Neisseria haemolysans but was reclassi¢ed into a new genus, Gemella, following demonstration of biochemical differences with other Neisseria. This species is easily decolorized during Gram staining and may therefore appear as Gram-variable or even Gram-negative. It can be misidenti¢ed as a viridans streptococcus or remain unidenti¢ed. An accurate

identi¢cation can be made with commercial biochemical tests such as the API 20 STREP system. To the best of our knowledge, 15 cases of endocarditis due to G. hemolysans have been reported so far [2^14].The available information is shown in Table 1. Mean age was 53.2 years and ranged from 20 to 77 years. Of 16 patients, only two were women (12.5%). All but two cases occured in patients with underlying mitral or aortic valve disease (including prosthetic valves) and/or poor dentition or dental manipulation [5,10]. Infected valves were aortic and mitral, both of them with the same percentage (50%). The outcome was completely favorable after antibiotic treatment, although some patients needed valve replacement [2^4,9,10,12,13]. In our patient, aortic valve insu¤ciency was the only risk factor identi¢ed for endocarditis but, interestingly, he had hemochromatosis.The association of G. haemolysans with hemochromatosis has not been reported to date and in this case probably represents a casual relationship between two uncommon conditions. However, the increased availability of iron in patients could have enhanced the virulence of G. haemolysans as has been demonstrated with other bacterial species such as Yersinia spp., Vibrio

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Clinical Microbiology and Infection, Volume 6 Number 10, October 2000

spp., and some mycobacteria among others [15]. In summary, our case suggests that hemochromatosis may be a risk factor for endocarditis due to G. haemolysans.

ACKNOWLEDGMENTS The authors wish to thank Drs J. Berenguer and E. Cercenado for their commentaries and their valuable assistance. J. D. Mosquera1, M. Zabalza1,M. Lantero2 andJ. R. Blanco1 1 Internal Medicine and 2 Microbiology Services, Complejo Hospitalario San Millan ^ San Pedro, Logron¬o, La Rioja, Spain Tel: ‡34 941 294500 E-mail: [email protected]

REFERENCES 1. Thjo«tta Th, Bo«e J. Neisseria haemolysans. A hemolytic species of Neisseria Trevisan. Acta Pathol Microbiol Scand Suppl 1938; 37: 527^ 31. 2. Chatelain R, Croize J, Rouge P et al. Isolement de Gemella haemolysans dans trios cas d'e ndocardites bacte¨ riennes. Med Mal Infect 1982; 1: 25^30. 3. Laudat P, Cosnay P, Icole B, Raoult A, Raynaud P, Brochier M. Endocardite a© Gemella haemolysans: une nouvelle observation. Med Mal Infect 1984; 4: 159^61. 4. Morea P,Toni M, Bressan M, Stritoni P. Prosthetic valve endocarditis by Gemella haemolysans. Infection 1991; 19: 446. 5. Kaufhold A, Franzen D, Lu«tticken R. Endocarditis caused by Gemella haemolysans. Infection 1989; 17: 385^7. 6. Brack MJ, Avery PG, Hubner PH, Swann RA. Gemella haemolysans: a rare and unusual cause of infective endocarditis. Postgrad MedJ 1991; 67: 210^2. 7. Fresard A, Michel VP, Rueda X, Aubert G, Dorche G, Lucht F. Gemella haemolysans endocarditis. Clin Infect Dis 1993; 16: 586^7. 8. Helft G, Tabone X, Metzger JP, Vacheron A. Gemella haemolysans endocarditis with colonic carcinoma. EurJ Med 1993; 2: 369^70. 9. Devuyst O, Hainaut P, Gigi J, Wauters G. Rarity and potential severity of Gemella haemolysans endocarditis. Acta Clin Belg 1993; 48: 52^3. 10. Matsis PP, Easthorpe RN. Gemella haemolysans endocarditis. Aust N Z J Med 1994; 24: 417^8. 11. Samuel L, Bloom¢eld P. Gemella haemolysans prosthetic valve endocarditis. Postgrad MedJ 1995; 71: 188. 12. Casado JL, Caldero¨n C, Quereda C, Fortu¨ n J, Loza A. Endocarditis por Gemella haemolysans con formacio¨n de absceso perivalvular. Enferm Infecc Microbiol Clin 1995; 13: 127^8. 13. La Scola B, Raoult D. Molecular identi¢cation of Gemella species from three patients with endocarditis. J Clin Microbiol 1998; 36: 866^71. 14. Iglesias LA, Lo¨pez-Lopategui MC, Almagro F, Bersitain X,Vidal MA. Endocarditis por Gemella haemolysans. Enferm Infecc Microbiol Clin 1999; 17: 538^9. 15. Frederick P. Iron and infection. Clin Infect Dis1998; 27: 1367^8.

Unusual high-performance liquid chromatography pro®le of a strain of Mycobacterium avium Mycobacterium avium complex (MAC) is the most frequently detected non-tuberculous mycobacterium, not only in AIDS patients, where it is responsible for disseminated and localized infections, but also in non-immunocompromised subjects. Among the latter, it is frequently responsible for pulmonary disease in elderly people with various predisposing conditions, and for cervical lymphadenitis in the children [1]. Several di¡erent approaches can be applied for the identi¢cation of MAC: conventional tests reveal well-de¢ned biochemical and cultural features; highly speci¢c commercial DNA probes (AccuProbe, USA) react with a species-speci¢c 16S rRNA stretch; and ¢nally, high-performance liquid chromatography (HPLC) of cell wall mycolic acids provides an easily recognizable pattern characterized by three clusters of peaks (Figure 1b). Herein, we report the characterization of two strains isolated from sputum collected at 5-month intervals from the same immunocompetent elderly subject. Both isolates had the phenotypic features of MAC, and both reacted with AccuProbe M. avium, but not with AccuProbe M. intracellulare.

Figure 1 Mycolic acid patterns obtained by HPLC: (a) pro®le of the ®rst strain (atypical); (b) pro®le of the second strain (MACtypical). LMWIS and HMWIS are low and high molecular weight internal standards, respectively.

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intracellulare, and by the members of the so-called MAC intermediates or MAI-X group [5], which react with AccuProbe MAC, but not with either AccuProbe ^ M. avium or AccuProbe ^ M. intracellulare. A similar anomaly of HPLC pro¢les has been previously reported in a few isolates of mycobacteria reacting with the MAC probe but not with the species-speci¢c ones [6]; in that case it was, however, possible to establish genetic sequences unrelated to MAC and very close to M. simiae. In the present case, hypervariable regions A and B of 16S rDNA [7] yielded a perfect match with the signature sequences of M. avium for both isolates. This is therefore the ¢rst report of an M. avium strain in which the 16S rDNA species-speci¢c markers do not correlate with the chromatographic pattern typical of this species. C. Garzelli and N. Lari were ¢nancially supported by MURST (Progetto di Ricerca 1997 `Controllo della Patogenicita© Microbica') and, partly, by ISS (Programma Nazionale sull'AIDS 1997, Italy). E.Tortoli*, A. Bartoloni, C. Garzelli, N. Lari, F. Lavinia, A. Mantella and S. Emler *Laboratorio di Microbiologia eVirologia, Piastra dei Servizi, Ospedale di Careggi, 50134 Firenze, Italy Tel: ‡ 39 055 4279199 Fax: ‡ 39 055 4279292 E-mail: [email protected] Figure 2 PvuII-generated IS1245-based RFLP ®ngerprints: M, molecular markers (kbp); a, second strain; b, ®rst strain.

REFERENCES

The IS1245-based restriction fragment length polymorphism analysis [2,3] of both isolates yielded di¡erent patterns (Figure 2), thus demonstrating the double origin of the infection. Surprisingly, the HPLC pro¢les of the two strains were completely di¡erent (Figure 1): the one obtained from the second strain (b) is like that established for M. avium and for MAC in general [4], whereas the HPLC pattern of the ¢rst strain (a) is unique, never seen in our experience with over 180 MAC strains characterized with HPLC and AccuProbe, and never reported for M. avium. The above data therefore add to our present knowledge of the HPLC pro¢le of MAC; the same typical three-clustered pro¢le is shared by strains assigned, on the basis of hybridization with speci¢c AccuProbes, to the species M. avium or M.

1. Falkinham JO III. Epidemiology of Mycobacterium avium infections in the pre- and post-HIVera. Res Microbiol 1994; 145: 169^72. 2. Garzelli C, Lari N, Nguon B, Cavallini M, Pistello M, Falcone G. Comparison of three restriction endonucleases in IS1245-based RFLPtypingofMycobacteriumavium.JMedMicrobiol1997;46: 933^9. 3. Guerrero C, Bernasconi C, Burki D, Bodmer T,Telenti A. A novel insertionelementfromMycobacteriumavium,IS1245,is aspeci¢ctarget foranalysisofstrainrelatedness.JClinMicrobiol1995;33: 304^7. 4. Butler WR,Thibert L, Kilburn JO. Identi¢cation of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography. J Clin Microbiol 1992; 30: 2698^704. 5. Viljanen MK, Olkkonen L, Katila ML. Conventional identi¢cation characteristics, mycolate and fatty acid composition, and clinical signi¢cance of MAIX AccuProbe-positive isolates of Mycobacterium avium complex. J Clin Microbiol 1993; 31: 1376^8. 6. Tortoli E, Piersimoni C, Kirschner P et al. Characterization of mycobacterial isolates phylogenetically related to, but di¡erent from, Mycobacterium simiae. J Clin Microbiol 1997; 35: 697^702. 7. Kirschner P, Springer B, Vogel U et al. Genotypic identi¢cation of mycobacteria by nucleic acid sequence determination: report of a 2-year experience in a clinical laboratory. J Clin Microbiol 1993; 31: 2882^9.

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