Comparative in vitro activities of five quinolone antibiotics, including gemifloxacin, against clinical isolates

CONCISE COMMUNICATIONS Comparative in vitro activities of five quinolone antibiotics, including gemifloxacin, against clinical isolates N. GoÈnuÈlluÈ1, Z. AktasË2, M. SËalciogÆlu2, CË. Bal 2 and OÈ. AngÆ 3 1 Institute for Experimental Medical Research, University of Istanbul, Istanbul, 2Department of Microbiology and Clinical Microbiology, Istanbul Faculty of Medicine, University of Istanbul, 34390 CËapa, Istanbul, Turkey and 3Formerly at 2 

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The in vitro activities of cipro¯oxacin, o¯oxacin, nor¯oxacin, levo¯oxacin and gemi¯oxacin against 343 clinical isolates were compared. Gemi¯oxacin showed the greatest activity, with MIC90 values as low as 0.03±0.25 mg/L against Streptococcus pneumoniae, Haemophilus in¯uenzae, Moraxella catarrhalis, methicillin-susceptible Staphylococcus aureus and Klebsiella pneumoniae, while methicillin-resistant Staphylococcus aureus, Enterococcus spp., Pseudomonas spp., Acinetobacter spp., Escherichia coli and Enterobacter spp. strains exhibited low rates of susceptibility to all ®ve ¯uoroquinolones. Keywords Fluoroquinolones, gemi¯oxacin, MIC Accepted 12 June 2001

Clin Microbiol Infect 2001; 7: 499±503

Quinolone antibiotics have been widely used for the treatment of various infections, particularly during the last decade. There is an increasing trend of quinolone resistance among several groups of bacteria in Turkey. This study has the objective of comparing the in vitro activities of ®ve ¯uoroquinolones against major human pathogens. Among the study antibiotics, cipro¯oxacin, o¯oxacin and nor¯oxacin have been used for a long period of time for treatment in Turkey, in parallel with their use in other countries; levo¯oxacin has been in clinical use for 1 year, and gemi¯oxacin is not yet on the market. Thus, the activities of three established quinolones and one recent broadspectrum quinolone are compared with the activity of an extended-spectrum quinolone which is not yet in use. The strains studied were: 50 Staphylococcus aureus, 26 of which were methicillin resistant (MRSA); 25 Enteroccocus spp.; 50 Klebsiella pneumoniae, 26 (52%) of which were extended-spectrum b-lactamase (ESBL) positive; 25 Enterobacter spp.; 25 Escherichia coli, 10 (40%) of which were ESBL positive; 50 Pseudomonas spp., 45 of which were Pseudomonas aeruginosa; 25 Acinetobacter spp.; 48 Streptococcus pneumoniae, 23 of which were penicillin resistant, including three high-level and 20 lowlevel resistant strains and ®ve erythromycin-resistant strains; 25 Haemophilus in¯uenzae, two (8%) of which were b-lactamase positive; 20 Moraxella catarrhalis, including 18 (90%) b-lactamase-positive strains. Together, these made up a total of 343 isolates. The majority of the Staphylococcus aureus isolates, enteric and non-fermentative Gram-negative bacilli, were obtained from tracheal aspirates and urine samples from adult patients hospitalized in the intensive care and renal transplantation units.

Pseudomonas spp. and methicillin-susceptible Staphylococcus aureus (MSSA) strains also included isolates from children with cystic ®brosis. Streptococcus pneumoniae, H. in¯uenzae and M. catarrhalis isolates were from community-acquired respiratory infections. The antibiotics used in this study were cipro¯oxacin, o¯oxacin, nor¯oxacin (Fako, Istanbul, Turkey), levo¯oxacin (Hoechst, Istanbul, Turkey; Fako) and gemi¯oxacin (GlaxoSmithKline, Istanbul, Turkey). MICs were determined by the agar dilution method, and results were interpreted using the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS) [1,2]. Since the NCCLS has not yet stated any breakpoints for gemi¯oxacin, breakpoints proposed by the British Society for Antimicrobial Chemotherapy were used (for Enterobacteriaceae, staphylococci, enterococci, hemophili, pneumococci and Pseudomonas spp., the proposed MIC breakpoints are 0.5 mg/L for susceptible and 1 mg/L for resistant) [3]. The control strains were Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Streptococcus pneumoniae ATCC 49619. Mueller±Hinton agar (Oxoid, Hemakim, Istanbul, Turkey) was used as the test medium; 5% de®brinated sheep blood was added to the medium for Streptococcus pneumoniae. Haemophilus Test Medium was used for testing H. in¯uenzae. An inoculum of 104 CFU/spot was delivered by a multipoint inoculator (Mast Diagnostics, Istanbul, Turkey) onto the test media containing the quinolone antibiotics in a series of twofold dilutions. Plates were incubated at 35 8C for 16±20 h for rapidly growing organisms and for 20±24 h for H. in¯uenzae

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MIC (mg/L) Bacterial species (n) Streptococcus pneumoniae Penicillin susceptible (25)

ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 499±513

Penicillin resistant (23)

Staphylococcus aureus Methicillin susceptible (24)

Methicillin resistant (26)

Enterococcus spp. (25)

Haemophilus influenzae (25)

Moraxella catarrhalis (20)

Drug

50%

90%

Range

S

I

R

Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin

0.03 1 1 2 8 0.03 1 2 2 16

0.03 1 2 2 8 0.06 2 4 4 8

0.004^0.03 0.5^1 1^2 1^2 4^16 0.03^0.12 1^2 0.5^8 1^16 4 to 64

100 100 100 100 ^ 100 100 78.3 78.3 ^

^ ^ ^ ^ ^ ^ ^ 21.7 13 ^

^ ^ ^ ^ ^ ^ ^ ^ 8.7 ^

Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin

0.06 0.12 0.5 0.5 2 1 4 16 8 32 0.06 1 2 2 2 0.008 0.015 0.008 0.03 0.03 0.015 0.06 0.06 0.03 0.12

0.06 0.25 2 1 16 2 4 16 16 64 4 32 64 64 128 0.03 0.03 0.03 0.06 0.06 0.03 0.06 0.06 0.03 0.12

0.06 0.06^0.5 0.25^4 0.25^1 1^64 0.06^4 0.12^8 0.12^64 0.5^16 0.5 to 128 0.06^16 0.5^64 1to 128 1to 128 1to 128 0.004^0.03 0.008^0.03 0.004^0.03 0.015^0.06 0.015^0.06 0.002^0.03 0.004^0.06 0.004^0.06 0.008^0.06 0.015^0.25

100 100 75 100 79.1 3.8 15.4 7.7 3.8 7.7 60 60 40 ^ 60 100 100 100 100 ^ ^ ^ ^ ^ ^

^ ^ 16.7 ^ 4.2 ^ 76.9 7.7 7.7 ^ ^ ^ 20 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

^ ^ 8.3 ^ 16.7 96.2 7.7 84.6 88.5 92.3 40 40 40 ^ 40 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

500 Clinical Microbiology and Infection, Volume 7 Number 9, September 2001

Table 1 Susceptibility of 343 clinical isolates to five quinolones

ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 499±513

Escherichia coli (25)

Klebsiella pneumoniae (50)

Enterobacter spp. (25)

Pseudomonas spp. (50)

Acinetobacter spp. (25)

Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin Gemifloxacin Levofloxacin Ciprofloxacin Ofloxacin Norfloxacin

8 8 16 16 128 0.06 0.12 0.06 0.12 0.25 0.12 0.12 0.06 0.12 0.12 1 4 1 4 2 0.5 4 16 8 64

64 32 128 64 128 0.25 1 0.5 1 2 8 4 16 16 64 16 32 16 32 32 8 8 128 16 128

0.06 to 128 0.06^64 0.06 to 128 0.06 to 128 0.06 to 128 0.06^1 0.06^8 0.06^4 0.06^8 0.06^32 0.06^16 0.06^8 0.06^64 0.06^16 0.06 to 128 0.06^64 0.06^64 0.06^32 0.06 to 128 0.5 to 128 0.06^8 0.12^16 0.25 to 128 0.25^32 4 to 128

44 44 44 44 44 96 98 96 98 98 84 84 84 84 84 44 46 56 44 62 56 36 24 36 8

^ ^ ^ ^ ^ ^ ^ 2 ^ ^ ^ 12 ^ ^ ^ ^ 14 6 12 10 ^ 16 12 ^ 24

56 56 56 56 56 4 2 2 2 2 16 4 16 16 16 56 40 38 44 28 44 48 64 64 68

S, % susceptible; I, % intermediate; R, % resistant. Where there is no percentage given for any category, NCCLS or BSAC breakpoints have not been established.



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and Streptococcus pneumoniae. All test organisms were incubated in ambient air, except for H. in¯uenzae, which was incubated at 3±7% CO2. The in vitro activities of the ®ve test quinolones according to their MIC90 values are summarized in Table 1. Gemi¯oxacin, levo¯oxacin and o¯oxacin were the most active agents against MSSA, with a 100% susceptibility rate for each; gemi¯oxacin had the lowest MIC90 (0.06 mg/L). On the other hand, only 3.8% of the MRSA strains remained susceptible to gemi¯oxacin. Twenty-two (91.7%) of the 24 MSSA and four (15.4%) of the 26 MRSA strains were susceptible to cipro¯oxacin (MIC <4 mg/L). Against MRSA strains, gemi¯oxacin demonstrated lower MIC50s and MIC90s than the other quinolones. Enterococcus spp. isolates had approximately equal rates of susceptibility against all ®ve quinolones, although gemi¯oxacin had the lowest MIC50 and MIC90. All test quinolones, including gemi¯oxacin, had approximately equivalent activity among each group of the enteric bacilli. Escherichia coli isolates were the least susceptible to all ®ve quinolones, with a resistance rate of 56% against each. Against K. pneumoniae, gemi¯oxacin was at least two times as active as the other quinolones, although all were active. The activity of levo¯oxacin against Escherichia coli and Enterobacter spp. strains was two times that of gemi¯oxacin. Gemi¯oxacin and levo¯oxacin seemed to be more active against Acinetobacter spp., and gemi¯oxacin and cipro¯oxacin seemed to be more active against Pseudomonas spp. The activity of gemi¯oxacin was very high and similar to those of levo¯oxacin, cipro¯oxacin and o¯oxacin against H. in¯uenzae and M. catarrhalis isolates. Both penicillin-resistant and penicillin-susceptible Streptococcus pneumoniae strains were highly susceptible to gemi¯oxacin. The activity of gemi¯oxacin was 32 and 64 times those of levo¯oxacin and cipro¯oxacin, respectively, against penicillin-resistant pneumococci, according to the MIC90 values. The in vitro activities of ®ve quinolone antibiotics were compared in order to determine the necessity for alternative quinolones in Turkish patients. In recent years, the clinical ef®cacies of cipro¯oxacin and other ¯uoroquinolones against infections with Staphylococcus aureus have decreased. Data indicate that ¯uoroquinolone resistance is considerably higher among MRSA strains than among MSSA strains [4±7]. In our study, levo¯oxacin was the most active compound against MRSA. Among the Gram-negative organisms responsible for our cases of hospital-acquired pneumonia, K. pneumoniae, Enterobacter, Acinetobacter and P. aeruginosa strains predominated. Quinolone resistance among these species has been reported [8,9], and we also found high MICs for our test quinolones, particularly against Escherichia coli, Pseudomonas spp. and Acinetobacter spp. Gemi¯oxacin exhibited the best activity, among the enteric isolates, against K. pneumoniae.

One of the major clinical targets of the newer quinolones comprises respiratory pathogens, and some are offered for empirical use in these infections. The most notable target of these new antibiotics is penicillin-resistant Streptococcus pneumoniae [10,11]. Our penicillin-resistant pneumococci, with either high- or low-level resistance, were not susceptible to cipro¯oxacin or o¯oxacin, with MIC90s of 4 mg/L each [12,13]. The activity of gemi¯oxacin was 32 times that of levo¯oxacin against penicillin-susceptible and -resistant pneumococci, according to the MIC90 values. With the exception of isolates from special patient groups, such as cystic ®brosis patients or patients with underlying malignancies for which penicillin resistance of pneumococci has been reported to be higher [14], the average rate of highlevel penicillin resistance is still 10% in Turkey [15±17]. In addition to Streptococcus pneumoniae, H. in¯uenzae and M. catarrhalis are the other major agents of community-acquired pneumonia (CAP), and these two species are still susceptible to many groups of antibiotics all around the world and in our country. This underlines the fact that uncomplicated cases of CAP can still be treated with conventional and cheaper drugs in Turkey, and that the newer quinolones may be saved for complicated cases of CAP, to delay the development of resistance against them. For these serious cases, the use of the most potent respiratory antibiotic, which seems to be gemi¯oxacin in our study, would be appropriate. On the other hand, the newer quinolones, including gemi¯oxacin, may be good alternatives for non-respiratory Gram-negative infections against which the older members of the quinolone family have lost potency. ACK NOW L EDGME N T S This study was supported by the Research Fund of the University of Istanbul, Project number B-448/26042000, and was presented as a poster at the 7th Scienti®c Meeting of the European Society of Chemotherapy and Infectious Diseases on 1±3 June 2000 in Sorrento, Naples, Italy. R EFER E NCE S 1. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th edn. Approved Standard M7-A5. Wayne, Pa: National Committee for Clinical Laboratory Standards, 2000. 2. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing, 10th informational supplement, Aerobic dilution, M100-S10. Wayne, Pa: National Committee for Clinical Laboratory Standards, 2000. 3. Wise R, Andrews JM. The in-vitro activity and tentative breakpoint of gemifloxacin, a new fluoroquinolone. J Antimicrob Chemother 1999; 44: 679±88. 4. Appelbaum PC, Hunter PA. The fluoroquinolone antibacterials: past, present and future perspectives. Int J Antimicrob Agents 2000; 16: 5±15.

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Concise Communications

5. Schmitz FJ, Fluit AC, Milatovic D, Verhoef J, Heinz HP, Brisse S. In vitro potency of moxifloxacin, clinafloxacin and sitafloxacin against 248 genetically defined clinical isolates of Staphylococcus aureus. J Antimicrob Chemother 2000; 46: 109±13. 6. Jones ME, Visser MR, Klootwijk M, Heisig P, Verhoef J, Schmitz FJ. Comparative activities of clinafloxacin, grepafloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin and nonquinolones linezolid, quinupristin±dalfopristin, gentamicin, and vancomycin against clinical isolates of ciprofloxacinresistant and -susceptible Staphylococcus aureus strains. Antimicrob Agents Chemother 1999; 43: 421±3. 7. Fu KP, Lafredo SC, Foleno B et al. In vitro and in vivo antibacterial activities of levofloxacin (l-ofloxacin), an optically active ofloxacin. Antimicrob Agents Chemother 1992; 36: 860±6. È ztuÈrk S et al. A surveillance study of 8. GuÈnseren F, Mamikoglu L, O antimicrobial resistance of gram-negative bacteria isolated from intensive care units in eight hospitals in Turkey. J Antimicrob Chemother 1999; 43: 373±8. 9. Brisse S, Milatovic D, Fluit AC et al. Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Enterobacter aerogenes clinical isolates with alterations in gyrA and ParC proteins. Antimicrob Agents Chemother 1999; 43: 2051±5. 10. Hoban DJ, Bouchillon SK, Karlowsky JA et al. A comparative in vitro surveillance study of gemifloxacin activities against 2632 recent Streptococcus pneumoniae isolates from across Europe, North

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America, and South America. Antimicrob Agents Chemother 2000; 44: 3008±11. Jorgensen JH, Weigel LM, Swenson JM, Whitney CG, Ferraro MJ, Tenover FC. Activities of clinafloxacin, gatifloxacin, gemifloxacin, and trovafloxacin against recent clinical isolates of levofloxacin-resistant Streptococcus pneumoniae. Antimicrob Agents Chemother 2000; 44: 2962±8. Pong A, Thomson KS, Moland ES, Chartrand SA, Sanders CS. Activity of moxifloxacin against pathogens with decreased susceptibility to ciprofloxacin. J Antimicrob Chemother 1999; 44: 621±7. Davies TA, Pankuch GA, Dewasse BE, Jacobs MR, Appelbaum PC. In vitro development of resistance to five quinolones and amoxicillin±clavulanate in Streptococcus pneumoniae. Antimicrob Agents Chemother 1999; 43: 1177±82. GuÈr D, TuncËkanat F, Sener B, Kanra G, Akalin HE. Penicillin resistance in Streptococcus pneumoniae in Turkey. Eur J Clin Microbiol Infect Dis 1994; 13: 440±4. È ngen B, Kaygusuz A, O È zalp M, GuÈrler N, ToÈreci K. Penicillin O resistance in Streptococcus pneumoniae in Istanbul, Turkey. J Clin Microbiol Infect 1995; 1: 150. Sener B, GuÈnalp A. Trends in antimicrobial resistance of Streptococcus pneumoniae in children in a Turkish hospital. J Antimicrob Chemother 1998; 42: 381±4. GoÈnuÈlluÈ N, Berkiten R. Antimicrobial resistance of clinical isolates of Streptoccocus pneumoniae in Istanbul. Int J Antimicrob Agents 2000; 16: 77±8.

Activity of quinupristin^dalfopristin in invasive isolates of Streptococcus pneumoniae from Italy A. Pantosti1, F. D'Ambrosio1, E. Bordi2, A. Scotto d'Abusco1 and M. Del Grosso1 1

Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di SanitaÁ, Viale Regina Elena 299, 00161 Rome and Laboratorio di Analisi Cliniche e Microbiologiche, Istituto Nazionale per le Malattie Infettive Lazzaro Spallanzani, Rome, Italy 2



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Eighty-®ve recent isolates of Streptococcus pneumoniae from patients with invasive disease were examined for their susceptibility to erythromycin, clindamycin, penicillin and quinupristin-dalfopristin by E test. A novel duplex PCR assay was used to detect the presence of the erm(B) or mef (A) genes in all of the erythromycinresistant isolates. All of the strains tested were susceptible to the combination quinupristin±dalfopristin, regardless of their susceptibility to penicillin or to erythromycin. By duplex PCR, two-thirds of the erythromycin-resistant strains harbored erm, and one-third harbored mef. The activity of quinupristin± dalfopristin was not in¯uenced by the genetic determinant of erythromycin resistance. The in vitro susceptibility of S. pneumoniae to quinupristin±dalfopristin is promising for future use; however, it is important to monitor the possible emergence of resistance. Keywords Macrolides, quinupristin±dalfopristin, Streptococcus pneumoniae, streptogramins Accepted 10 May 2001

Clin Microbiol Infect 2001; 7: 503±506 IN T R ODUC T ION Antibiotic resistance in Streptococcus pneumoniae is becoming a worrying worldwide problem [1]. The pattern of Streptococcus

pneumoniae resistance in Italy is different from that of other European countries: resistance to penicillin is low or moderate and is mainly ascribed to strains with intermediate susceptibility to penicillin, while resistance to erythromycin has increased

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504 Clinical Microbiology and Infection, Volume 7 Number 9, September 2001

steadily in the last 5 years and is now present in over 30% of the isolates [2]. In addition, in the USA, the majority of erythromycin-resistant strains bear the mef (ef¯ux) gene and display an M phenotype, characterized by a low level of resistance to 14- and 15-membered macrolides only [3,4]. Conversely, in Italy the predominant resistance phenotype is MLSB, characterized by a high level of resistance to erythromycin and other macrolides, in association with resistance to lincosamides and streptogramin B, due to expression of the erm gene, which encodes for a methylase that modi®es the ribosomal target [2,5,6]. Quinupristin±dalfopristin (Synercid) is a new streptogramin antibiotic consisting of a combination of two structurally unrelated compounds, a streptogramin A (quinupristin) and a streptogramin B (dalfopristin), which act synergistically [7]. As the armamentarium of antibiotics available to treat pneumococcal infections is becoming smaller, it is becoming important to investigate the activity of new possible therapeutic options. We studied the activity of quinupristin±dalfopristin against a collection of recent invasive Streptococcus pneumoniae isolates, either susceptible or resistant to erythromycin, and determined their phenotypes and genotypes of resistance. ME T HOD S Eighty-®ve invasive Streptococcus pneumoniae isolates, 65 from cerebrospinal ¯uid (CSF) and 22 from blood, collected in Italy in the years 1997±99, were studied. The CSF isolates were obtained as part of the National Surveillance System of Bacterial Meningitis, a laboratory-based system involving the whole country. Most blood isolates were obtained from hospitals in the Rome area. All isolates sent to the Istituto Superiore di SanitaÁ in 1997±99 from these sources that were resistant to erythromycin and/or penicillin (including the penicillin-intermediate strains) were used for the study. The collection included 39 strains resistant to erythromycin, six strains resistant to penicillin, and 10 strains resistant to both erythromycin and penicillin. In addition, 30 strains susceptible to both antibiotics

were randomly chosen from these same sources. Minimal inhibitory concentrations (MICs) of erythromycin, clindamycin, penicillin and quinupristin±dalfopristin were determined by E test (AB Biodisk, Solna, Sweden), following the manufacturer's recommendations. The suspension of bacterial cells, at a density corresponding to 0.5±1 McFarland units, was swabbed onto Mueller±Hinton II agar (Beckton-Dickinson Italia, Milan, Italy) plates supplemented with 5% sheep blood. The reference strain ATCC 49619 was tested as internal control every time the susceptibility assay was performed. If the MIC obtained with the E test fell between the standard two-fold increments, it was rounded to the next higher log2. The breakpoints used were those de®ned by the National Committee for Clinical Laboratory Standards (NCCLS) [8]. All the erythromycin-resistant isolates were subjected to a duplex PCR assay to detect the presence of erm(B) or mef (A) [9]. The two primer pairs used, derived from Sutcliffe [10] with minor modi®cations, were: EB1 (50 -GAAAAAGTA CTCAACCAAATA-30 ) and EB2 (50 -AGTAATGGTACTTAAATTGTTTAC-30 ) to amplify erm(B); and ME1 (50 -AGTA TCATTAATCACTAGTGC-30 ) and ME2 (50 -TTCTTCTG GTACTAAAAGTGGGC-30 ) to amplify mef (A). Boiled pneumococcal bacterial cells were used as template. The reaction mixture contained 50 pmol each of EM1 and EM2, 15 pmol each of ME1 and ME2, 100 mM each deoxynucleoside triphosphate, 1 U of Dynazyme II DNA polymerase (Finnzyme, Oy, Finland), and 3 mM MgCl2, in a ®nal volume of 50 mL. Samples were subjected to 35 ampli®cation cycles with an annealing temperature of 50 8C. Ampli®ed DNA was analyzed by gel electrophoresis. The predicted ampli®cation product for the erm gene was approximately 640 bp, and that for the mef gene was approximately 350 bp. R E S ULT S The susceptibilities of the pneumococcal strains tested are summarized in Table 1. All strains were susceptible to the combination quinupristin±dalfopristin, regardless of their

Table 1 Susceptibilities of 85 invasive Streptococcus pneumoniae strains to quinupristin^dalfopristin and other antibiotics MIC (mg/L) Strains (no.)

Antibiotic

Range

MIC50

MIC90

% Susceptible

Erythromycin susceptible (36) (including 6 penicillin non-susceptible)

Erythromycin Clindamycin Penicillin Quinupristin^dalfopristin Erythromycin Clindamycin Penicillin Quinupristin^dalfopristin

0.06^0.25 0.06^0.25  0.015^2 0.5^1 4 to  256 0.03 to  256  0.015^2 0.5^1

0.12 0.12  0.015 0.5  256  256 0.03 1

0.12 0.12 1 1  256  256 0.12 1

100 100 84 100 0 31 80 100

Erythromycin resistant (49) (including 10 penicillin non-susceptible)

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DI SC US S ION

Figure 1 Gel electrophoresis (1.2% agarose) of the products obtained by a duplex PCR assay to detect mef (A) and erm(B). The amplicons erm and mef are, respectively, 640 and 350 bp. M, molecular mass marker. Lane 1: PCR product obtained using a mixture of the chromosomal DNAs of an erm-containing strain and a mef-containing strain. Lane 2: strain positive for mef. Lanes 3^5: strains positive for erm.

susceptibility to erythromycin or to penicillin. However, the MICs of the strains fell within a very narrow range (0.5±1 mg/L) that was near or at the breakpoint for susceptibility, established by the NCCLS at 1 mg/L [8]. All the erythromycin-sensitive strains were also sensitive to clindamycin, while only 15 of 49 (30%) erythromycin-resistant strains were clindamycin susceptible (Table 1). These 15 strains exhibited an M phenotype and had MICs for erythromycin between 4 and 64 mg/L. The other 34 erythromycin-resistant strains were resistant to high levels of both erythromycin and clindamycin, and therefore exhibited an MLSB phenotype. With duplex PCR (Figure 1), all of the former group of strains were found to harbor mef(A), and the latter group erm(B). No strain harboring both genes was found. This distribution of erm and mef, with approximately two-thirds of the erythromycin-resistant strains harboring erm, and one-third harboring mef, appears to be typical among Italian invasive isolates [2]. In Italian respiratory isolates, however, strains exhibiting an MLSB phenotype or carrying erm are predominant, representing 90±95% of the erythromycin-resistant isolates [5,6]. Although the presence of erm(B) could affect the activity of dalfopristin, which is a streptogramin B, no difference was noted in the activity of the combination between strains harboring erm and strains harboring mef (not shown).

This study demonstrates that the combination quinupristin± dalfopristin is fully active against invasive Streptococcus pneumoniae strains isolated in Italy, including strains highly resistant to erythromycin. This result essentially con®rms and extends those of previous reports, in that the activity of quinupristin± dalfopristin is independent of resistance to other antibiotics, including those that have a common target [11±13]. Although dalfopristin is a streptogramin B compound, the combination of the two components retains its synergistic activity against bacteria with the MLSB phenotype carrying the erm methylase gene. This ®nding is particularly relevant for the potential use of the antibiotic combination in Italy, where erythromycin resistance in Streptococcus pneumoniae is very frequent and resistance is conferred by the erm gene in at least 70% of the strains [2], as con®rmed in our study using a duplex PCR assay. Although in our study all strains were susceptible to quinupristin±dalfopristin, their MICs were very close to the upper limit for the susceptible category. In contrast to other reports [11,12,14], in our strains we never found an MIC lower than 0.5 mg/L: 0.38 mg/L was the lowest non-corrected MIC obtained from the E test strip, a level 2±4-fold higher than in other studies. This might be ascribed, at least in part, to the fact that we used E test instead of the reference microbroth dilution. However, the MIC50 and MIC90 were similar to those obtained in other studies [11,12,14±16], and the E test result for the control strain, ATCC 49619, was consistently within the acceptable range. Therefore, it is possible that a shift towards higher MIC has already happened in the Italian pneumococcal isolates, with the disappearance of the very susceptible isolates. This could be of concern, because failure of streptogramin treatment in pneumococcal pneumonia due to an isolate with an MIC of 1 mg/L has been reported [17]. However, in the reported case, treatment was with pristinamycin, whose plasma levels are below 1 mg/L with standard dosage [17]. Quinupristin±dalfopristin serum levels normally reach 2±3 mg/L [18] and therefore appear suf®cient to inhibit microorganisms with an MIC of 1 mg/L. In spite of the uniform in vitro susceptibility of Streptococcus pneumoniae to quinupristin±dalfopristin regardless of whether the strains were erythromycin susceptible or resistant, Reinert et al. [13] found a different pro®le of the time±kill curves. In erythromycin-resistant strains, the killing time was delayed, and, more importantly, there was regrowth after 3 h at a concentration of quinupristin±dalfopristin three times the MIC. A discrepancy between MIC determination and time±kill results in macrolide-resistant Streptococcus pneumoniae strains has been shown also for pristinamycin, another synergistic combination of two streptogramins [19]. Therefore, it is unclear whether quinupristin±dalfopristin is able to kill erythromycin-resistant

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506 Clinical Microbiology and Infection, Volume 7 Number 9, September 2001

microorganisms at the site of infection. Tarasi et al. [20] found that quinupristin±dalfopristin can penetrate the meningeal walls at the time of in¯ammation in a rabbit model, and reduce the number of infecting microorganisms by several logs. However, the strain used in this study was fully sensitive to most antibiotics, and more evidence is needed to support the use of this antibiotic combination for invasive Streptococcus pneumoniae infections. In vitro resistance to streptogramin antibiotics has been reported in other Gram-positive microorganisms. Resistance is linked to genes either inactivating [21] or extruding [22] the streptogramin A component from the bacterial cell. In the USA and some European countries, quinupristin± dalfopristin has been licensed for the treatment of serious infections caused by multiresistant Enterococcus faecium and Staphylococcus aureus. In the future, it will be important to monitor the susceptibility of microorganisms, including Streptococcus pneumoniae, to quinupristin±dalfopristin following its use in humans. ACK NOW L EDGME N T S This study was supported in part by grants from Ministero della SanitaÁ, Programmi per la Ricerca Finalizzata 1998, and from Progetti di ricerca ®nalizzata IRCCS, ICS 120.5/RF 97.99. R EFER E NCE S 1. Tomasz A. New faces of an old pathogen: emergence and spread of multidrug-resistant Streptococcus pneumoniae. Am J Med 1999; 107(1A): 55S±62S. 2. Pantosti A, D'Ambrosio F, Tarasi A, Recchia S, Orefici G, Mastrantonio P. Antibiotic susceptibility and serotype distribution of Streptococcus pneumoniae causing meningitis in Italy, 1997±99. Clin Infect Dis 2000; 31: 1373±9. 3. Shortridge VD, Doern GV, Brueggemann AB, Beyer JM, Flamm RK. Prevalence of macrolide resistance mechanisms in Streptococcus pneumoniae isolates from a multicenter antibiotic resistance surveillance study conducted in the United States in 1994±95. Clin Infect Dis 1999; 29: 1186±8. 4. Gay K, Baughman W, Miller Y et al. The emergence of Streptococcus pneumoniae resistant to macrolide antimicrobial agents: a 6-year population-based assessment. J Infect Dis 2000; 182: 1417±24. 5. Marchese A, Tonoli E, Debbia EA, Schito GC. Macrolide resistance mechanisms and expression of phenotypes among Streptococcus pneumoniae circulating in Italy. J Antimicrob Chemother 1999; 44: 461±4. 6. Oster P, Zanchi A, Cresti S et al. Patterns of macrolide resistance determinants among community-acquired Streptococcus pneumoniae isolates over a 5-year period of decreased macrolide susceptibility rates. Antimicrob Agents Chemother 1999; 43: 2510±12.

7. Leclercq R, Courvalin P. Streptogramins: an answer to antibiotic resistance in Gram-positive bacteria. Lancet 1998; 352: 591±2. 8. National Committee for Clinical Laboratory Standarda (NCCLS). Performance standards for antimicrobial susceptibility testing; ninth informational supplement. M100-S9. Wayne, Pa: NCCLS, 1999. 9. Roberts MC, Sutcliff J, Courvalin P, Jensen LB, Rood J, Seppala H. Nomenclature for macrolide and macrolide±lincosamide± streptogramin B resistance determinants. Antimicrob Agents Chemother 1999; 43: 2823±30. 10. Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 1996; 40: 2562±6. 11. Barry AL, Fuchs PC, Brown SD. Antipneumococcal activities of a ketolide (HMR 3647), a streptogramin (quinupristin±dalfopristin), a macrolide (erythromycin) and a lincosamide (clindamycin). Antimicrob Agents Chemother 1998; 42: 945±6. 12. Betriu C, Redondo M, Palau ML et al. Comparative in vitro activities of linezolid, quinupristin±dalfopristin, moxifloxacin and trovafloxacin against erythromycin-susceptible and -resistant streptococci. Antimicrob Agents Chemother 2000; 44: 1838±41. 13. Reinert RR, Kresken M, Mechery V, Lemperle M, LuÈtticken R. In vitro activity of quinupristin/dalfopristin against erythromycinsusceptible and erythromycin-resistant Streptococcus pneumoniae. Eur J Clin Microb Infect Dis 1998; 17: 662±5. 14. Schouten MA, Hoogkamp-Korstanje JAA. Comparative in vitro activities of quinupristin±dalfopristin against Gram-positive bloodstream isolates. J Antimicrob Chemother 1997; 40: 213±19. 15. Dowzicky M. Evaluation of in vitro activity of quinupristin/ dalfopristin and comparator antimicrobial agents against worldwide clinical trial and other laboratory isolates. Am J Med 1998; 104(5A): 34S±42S. 16. Schmitz F-J, Verhoef J, Fluit AC, The Sentry Participants Group. Prevalence of resistance to MLS antibiotics in 20 European university hospitals participating in the European SENTRY surveillance programme. J Antimicrob Chemother 1999; 43: 783±92. 17. Burucoa C, Pasdeloup T, Chapon C, FaucheÁre JL, Robert R. Failure of pristinamycin treatment in a case of pneumococcal pneumonia. Eur J Clin Microbiol Infect Dis 1995; 14: 341±2. 18. Johnson CA, Taylor CA III, Zimmerman SW et al. Pharmacokinetics of quinupristin±dalfopristin in continuous ambulatory peritoneal dialysis patients. Antimicrob Agents Chemother 1999; 43: 152±6. 19. Schlegel L, Sissia G, FreÂmaux A, Geslin P. Diminished killing of pneumococci by pristinamycin demonstrated by time±kill studies. Antimicrob Agents Chemother 1999; 43: 2099±100. 20. Tarasi A, Dever LL, Tomasz A. Activity of quinupristin/ dalfopristin against Streptococcus pneumoniae in vitro and in vivo in the rabbit model of experimental meningitis. J Antimicrob Chemother 1997; 39(suppl A): 121±7. 21. Acar J, Casewell M, Freeman J, Friis C, Goosens H. Avoparcin and virginiamycin as animal growth promoters: a plea for science in decision-making. Clin Microbiol Infect 2000; 6: 477±82. 22. Werner G, Witte W. Characterization of a new enterococcal gene satG, encoding a putative acetyltransferase conferring resistance to streptogramin A compound. Antimicrob Agents Chemother 1999; 43: 1813±14.

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Persistent erythrovirus B19 urinary tract infection in an HIV-positive patient L. S. Christensen1, T. V. Madsen1 and T. Barfod2 Departments of 1Clinical Microbiology and 2Infectious Diseases, Rigshospitalet, DK-2100 Copenhagen East, Denmark 

Tel: ‡45 35 454974

Fax: ‡45 35 456412 E-mail: [email protected]

We report a urinary tract infection (UTI) with erythrovirus B19 in an HIV-1-positive homosexual man persisting for more than 7 months after the decline of viremia after a primary infection. During the course of the UTI, the patient complained of soreness in the kidney region and suffered from transient episodes of edema and hematuria. Proteinuria and elevated serum concentrations of creatinine further substantiated the hypothesis of a renal focus of a persistent erythrovirus B19 infection. Keywords Erythrovirus B19, parvovirus, urinary tract infection, HIV Accepted 11 May 2001

Clin Microbiol Infect 2001; 7: 507±509

Erythrovirus B19 (previously parvovirus B19) is the cause of a wide variety of clinical manifestations, including erythema infectiosum (®fth disease) in children and young adults, acute and persistent arthropathy in adults, fetal hydrops and other fetal complications if acquired during pregnancy, transient aplastic crisis, and chronic anemia in patients with hematologically predisposing conditions [1]. The variety of clinical manifestations is assumed to re¯ect the tropism of B19 to tissues expressing the viral receptor (erythrocyte P antigen), such as erythrocytes, megakaryocytes, endothelium, placenta, fetal liver, and myocardium. Erythrovirus B19 viremia is normally detectable by PCR up to 2 months after a primary infection [2], but persistent viremic infections have been reported in HIVinfected individuals [3], and persistence has been demonstrated also in synovial membranes of patients with and without chronic arthropathy [4]. In the present study, we report a case of an HIV-1-positive homosexual man with an erythrovirus B19 urinary tract infection (UTI) persisting for more than 7 months after the decline of the viremia and developing symptoms of renal involvement. A 39-year-old homosexual HIV-positive (CD4, 200/mm3; HIV RNA, <200/mL) male with an idiopathic chronic hepatocellular in¯ammation developed a maculopapulous rash on chest and legs following a 1-week-long period of fever. No joint symptoms or symptoms of renal involvement were noticed. A suspicion of an acute erythrovirus B19 infection, supported by a major epidemic in Denmark at the time, was con®rmed by the EIA detection of B19 IgM (Biotrin Ltd, Dublin, Ireland) and B19 DNA by PCR according

to Hornsleth et al. [5]. B19 IgG was detectable by EIA (Biotrin Ltd, Ireland) 2 months later. Eight weeks after the initial diagnosis of a B19 infection, a slight rash was still visible, and dysuria was reported for the ®rst time as well as a proteinuria (8 mmol/L). No hepatosplenomegaly or soreness of the kidney region had been noticed at the time. At weeks 19, 31 and 43, progressive fatigue was reported, with plasma levels of iron, transferrin and thyroid stimulating hormone (TSH) found to be normal. At week 66, soreness of the kidney region, hematuria, urine incontinence, edema and a slightly elevated serum level of creatinine were reported. X-ray investigations revealed no kidney or bladder stones, but cystoscopic investigation at week 66 showed a thickening of the bladder wall. Pathologic investigation of a bladder wall biopsy taken at week 96 revealed no cytopathic effect. Hematuria, as well as slightly elevated serum levels of creatinine, were reported at week 86 and again at week 96. A proteinuria (4 mmol/L) was demonstrated, while analyses of urine for the presence of cytomegalovirus, adenovirus, and BK virus by PCR, and bacteria by cultivation on blood agar and lactose±McConkey agar plates, were negative. A marked drop in hemoglobin concentration to 7.4 mmol/L was recorded at the time of diagnosis of the B19 infection. Otherwise, the hemoglobin concentrations were normal (9.0±10.1 mmol/L) throughout the study period. CD4 cell counts increased from 130 to 420 per mm3, and quantitative values of HIV-1 RNA were below 200/mL throughout the study period, presumably because of adequate highly active anti-retroviral treatment (HAART) treatment. No treatment of the B19 infection was

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508 Clinical Microbiology and Infection, Volume 7 Number 9, September 2001

Figure 1 Summary of laboratory findings.

attempted, yet all urogenital symptoms had resolved 2 years after the initial symptoms of a B19 infection. Incidentally, we requested urine samples from this and from other patients infected with erythrovirus B19 in order to evaluate the feasibility of using urine for this diagnosis. A bladder wall biopsy was provided at week 96, while no renal or bone marrow biopsy was provided. The laboratory ®ndings for a 2-year period are summarised in Figure 1, and the result of a sequence analysis is shown in Figure 2. Erythrovirus B19 DNA can be detected in urine samples from acutely infected children and adults infrequently and only for short periods (unpublished data), but persistent shedding of erythrovirus B19 in urine is reported here for the ®rst time. Laboratory data revealed a primary infection of erythrovirus B19 and a subsequent normal antibody response and decline of viremia. The decline in viremia and persistently high concentration of virus in urine strongly indicate a urinary tract focus of viral replication. The detection of B19 DNA in a bladder wall biopsy after the decline of the viral concentration in urine indicates a latent/persistent infection, possibly in the bladder epithelium or in the kidneys, where the endothelial cells of the glomeruli are candidate host cells expressing the viral receptor. Although a causal relationship could not be well established in this case, the renal effects make the latter of the two possibilities the most likely one.

It is remarkable that the erythrovirus B19 epidemic in Denmark in 1997 was caused by strains revealing a sequence heterogeneity comparable to the heterogeneity of globally circulating strains, suggesting that B19 epidemics are composed of a variety of strains. The sequence markers establish the relationship of the UTI strain with strains circulating in the same area at the time, as indicated in Figure 2. The UTI strain, like the other Danish strains, reveals a small number of unique mutations, making each of the strains unique. These mutations in the UTI strain were synonymous and therefore do not substantiate the hypothesis of a selective advantage of this particular strain. Glomerulonephritis has previously been reported in patients with sickle cell anemia who developed classic features of nephritic syndrome shortly after a B19-mediated transient aplastic crisis [6], and most recently in an adult immunocompetent host [7]. In these cases, B19 antigen could not be detected after biopsy, and a pathogenesis of glomerular damage from B19-associated immune complexes, or alternatively a microscopic vasculitis resembling the changes of polyarteritis nodosa, were suggested. Our data on persistent shedding of B19 in urine warrants further investigation into an alternative pathogenesis of a possibly rare, non-sickle cell disease-related nephritic syndrome preceded by high activity of renal replication of B19.

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ACK NOW L EDGME N T S We would like to thank Rikke Jùnson and Lis Nielsen for technical assistance, and Rigshospitalet, the Danish Medical Research Council (grant no. 12-1667) and the Novo Nordisk Foundation for ®nancial support. R EFER E NCE S

Figure 2 Dendrogram (Clustal X) based on nucleotides 2647^3941 of Shade et al. [8], including strains of erythrovirus 19 from the 1997 epidemic in Denmark and representatives of globally circulating strains [9]. All bootstrap values above 50% are indicated. The Danish strains are named DEN followed by week and year of isolation. DEN/13.97/EP is the UTI strain.

1. Brown KE, Young NS, Liu JM. Molecular, cellular and clinical aspects of parvovirus B19 infection. Crit Rev Oncol Hematol 1994; 16: 1±31. 2. Erdman DE, Usher MJ, Tsou C et al. Human parvovirus B19 specific IgG, IgA, and IgM antibodies and DNA in serum specimens from persons with erythema infectiosum. J Med Virol 1991; 35: 110±15. 3. Frickhofen N, Abkowitz JL, Safford M et al. Persistent B19 parvovirus infection in patients infected with human immunodeficiency virus type 1 (HIV-1): a treatable cause of anemia in AIDS. Ann Intern Med 1990; 113(12): 926±33. 4. SoÈderlund M, von Essen R, Haapasaari J, Kiistala U, Kiviluoto O, Hedman K. Persistence of parvovirus B19 DNA in synovial membranes of young patients with and without chronic arthropathy. Lancet 1997; 349: 1063±5. 5. Hornsleth A, Carlsen KM, Christensen LS, Gundestrup M, Heegaard ED, Myhre J. Estimation of serum concentrations of parvovirus B19 DNA by PCR in patients with chronic anaemia. Res Virol 1994; 145: 379±86. 6. Wierenga KJJ, Pattison JR, Brink N et al. Glomerulonephritis after human parvovirus infection in homozygous sickle-cell disease. Lancet 1995; 34: 475±6. 7. Taylor G, Drachenberg C, Faris-Young S. Renal involvement of human parvovirus B19 in an immunocompetent host. CID 2001; 32: 167±9. 8. Shade RO, Blundell MC, Cotmore SF, Tattersall P, Astell CR. Nucleotide sequence and genome organisation of human parvovirus B19 isolated from the serum of a child during aplastic crisis. J Virol 1986; 58: 921±36. 9. Erdman DD, Durigon EL, Wang Q-Y, Anderson LJ. Genetic diversity of human parvovirus B19: sequence analysis of the VP1/ VP2 gene from multiple isolates. J Gen Virol 1996; 77: 2767±74.

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510 Clinical Microbiology and Infection, Volume 7 Number 9, September 2001

Campylobacter fetus pericarditis in a patient with b-thalassemia: case report and review of the literature S. S. Kanj1, G. F. Araj3, A. Taher2 and L. B. Reller4 1

Division of Infectious Diseases and 2Division of Haematology, Department of Internal Medicine, American University of Beirut, PO Box 113-6044, Beirut, Lebanon, 3Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon and 4Department of Pathology, Duke University Medical Center, Durham, NC, USA 

Tel: ‡11 961 1 350 000

Fax: ‡11 961 1 370 814 E-mail: [email protected]

A case of pericardial effusion due to Campylobacter fetus in a patient with thalassemia is presented. The patient failed to respond to ceftriaxone and clarithromycin despite in vitro susceptibility, but improved after pericardiectomy and ampicillin. Pericarditis due to C. fetus has rarely been reported. A high index of suspicion is essential to recognise this organism, because of its special microbiological characteristics. Keywords Pericarditis, tamponade, Campylobacter fetus, thalassemia Accepted 11 May 2001

Clin Microbiol Infect 2001; 7: 510±513

C A S E R E P OR T The patient is a 14-year-old girl with b-thalassemia after major splenectomy. She had received all the childhood vaccinations and had no previous episodes of bacterial infections. On 30 March, she complained of fever, chills, vomiting and diarrhea. On 2 April, she presented to the American University of Beirut Medical Center. She denied any intake of unpasteurised milk products or raw meat, and exposure to farm animals. She had a temperature of 38.6 8C. A chest X-ray (CXR) revealed an enlarged heart, with a right pleural effusion. She was given 1 g of ceftriaxone and was sent home on cefuroxime. She returned the following day with severe dyspnea and dry cough. Her temperature was 40 8C, the respiratory rate was 24/min, the heart rate was 153/min, and the blood pressure was 100/ 50 mmHg. Heart examination showed distant sounds with a pericardial friction rub. The lungs had decreased air entry bilaterally at the bases with crackles. The peripheral white blood cell (WBC) count was 39  109/L, with 88% neutrophils. Arterial blood gas showed a PaO2 of 71 and oxygen saturation of 94%. An electrocardiogram revealed diffuse ST segment elevation. CXR showed enlargement of the cardiac silhouette with pulmonary congestion. Pericardial effusion was suspected, and the patient was admitted. Blood was taken for culture. An echocardiogram revealed a large pericardial effusion, and mild mitral regurgitation with signi®cant respiratory variation across the mitral and the tricuspid valves, indicating tamponade. A percutaneous pericardiocentesis yielded 300 mL of ¯uid. A small catheter was left in. The patient was started on ceftriaxone and vancomycin. The WBC count on the pericardial ¯uid was

34  103/mm3, with 97% neutrophils, glucose was 10 mg/dL, and protein 59 g/L. The Gram stain was negative. The patient remained dyspneic and febrile. On 6 April, she underwent anterior pericardiectomy. Fibrinous material was removed from around the epicardiac surface. The pathology revealed acute in¯ammation and ®brinopurulent exudate (Figure 1). The pericardial ¯uid grew pleomorphic curved Gram-negative rods, giving positive reactions for oxidase and catalase, and positive motility tests, and preferentially growing under 5±7% CO2. This isolate was identi®ed using API Campy ( bioMerieux, Marcy-L'Etoile, France) as Campylobacter fetus. Clarithromycin was added. A repeat echocardiogram showed constrictive pericarditis. The patient remained febrile. In vitro susceptibility testing using E test (PDM-Epsilometer, AB Biodisk, Solna, Sweden) revealed the organism to be susceptible to ampicillin, cephalothin, cefotaxime, erythromycin, chloramphenicol, cipro¯oxacin, clindamycin, gentamicin, tetracycline, imipenem, and trimethoprim±sulfamethoxazole. It was resistant to cefuroxime, ceftazidime, piperacillin±tazobactam, and aztreonam. Ceftriaxone and clarithromycin were stopped, and the patient was started on 8 g daily of ampicillin on 14 April. Twenty-four hours later, the patient defervesced. A repeat echocardiogram revealed that the signs of constrictive pericarditis had improved. After 21 days, the patient was discharged home on amoxicillin 3 g daily. Upon follow-up in the clinic 15 days later, the patient was asymptomatic. Ampicillin was discontinued 3 weeks later. Because of the dif®culties encountered in the recovery and the de®nitive identi®cation of the microorganism, the isolate was referred to the Microbiology Laboratory at Duke

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Figure 1 Pericardial biopsy showing fibrinopurulent exudate containing neutrophils.

University Medical Center and was con®rmed to be C. fetus subsp. fetus. DI SCUS S ION To our knowledge, this is the ®rst case of C. fetus pericarditis to be reported in a patient with thalassemia. Patients with b-thalassemia are more prone to develop several types of infections, including transfusion-associated viral infections such as hepatitis B and C [1], and certain bacterial infections such as Yersinia enterolitica infection [2]. Moreover, patients who have undergone splenectomy are at a higher risk of developing infections with the encapsulated bacterial organisms. However, there are no previous reports on any association between thalassemia and/or splenectomy and infection with C. fetus. One previous report described a fatal infection with C. jejuni in a post-splenectomy thalassemic patient who presented with fever and rapidly progressed to septicemic shock [3]. C. fetus has been known to cause abortion in domestic animals since 1909 [4]. Occasionally, the organism causes infection in humans, being ®rst recovered from uterine discharges in 1913 [5]. In 1970, Bokkenhouser et al. published 10 cases of infection with Vibrio fetus isolated from the blood, the cerebrospinal ¯uid, and abscesses [6]. Most of the affected patients had underlying chronic illnesses, which led to this organism being considered as an opportunistic pathogen [7,8]. V. fetus differed in DNA composition from the other Vibrio species, so a new genus (Campylobacter) was proposed in 1972 by SeÂbald and Veron [9]. C. jejuni and C. fetus were recognised as human pathogens, whereas some of the other species were only occasionally incriminated in human diseases.

The main mechanisms of transmission of campylobacter infection to humans have been: (1) direct contact with infected animals, although most patients with documented infection have no such history [10]; and (2) through contaminated milk or food Ð epidemics of campylobacter infection have been reported following ingestion of raw liver and milk [11,12]. In our patient, the exact route of infection is not known. Possibly, it might have originated in the gastrointestinal tract, since the patient reported diarrhea, which could have resulted in bacteremia and seeding of the pericardium. However, no stool cultures were obtained. Moreover, a previous study from Lebanon revealed a low prevalence of Campylobacter in human diarrheic stools [13]. C. fetus infection has been reported in several clinical settings, including: bacteremia, septic abortions, meningoencephalitis, motor neuron paralysis, brain abscesses, subdural empyema, septic arthritis, vertebral osteomyelitis, lung abscesses, peritonitis, and acute cholecystitis [6±8]. Extraintestinal spread of C. fetus has been associated with surface-layer proteins that mediate both complement resistance and antigenic variation in mammalian hosts. Site-speci®c reciprocal recombination between genes leads to expression of divergent surface-layer proteins as one of the mechanisms that C. fetus uses for antigenic variation that could explain the extraintestinal spread [14]. This organism has a predilection for the cardiovascular system, with involvement of the vascular endothelium, especially in the presence of pre-existing vascular damage. Possible theories to explain this include the ability of the organism to produce a local procoagulant that promotes thrombus formation or the presence of a surface receptor with a high af®nity for the endothelium, leading to endothelial damage and thrombus formation [15]. Cardiovascular manifestations reported in the

ß 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7, 499±513

Death

Recovery

Recovery

Recovery Recovery

Blood and pericardium None

61

74

[23]

[24]

Hypertension Diabetes mellitus Mitral stenosis

None

Pericardium and blood Blood Agriculture 52 [22]

Alcoholism

None None 57 36 [20] [21]

Hypothyroidism Polycystic kidney disease

None 60 [19]

Lymphoma

48 [18]

Rheumatic fever

Lived on a farm

Blood Pericardium Blood Pericardium (post mortem) Pericardium Blood

Ampicillin Ampicillin Erythromycin Doxycycline Erythromycin Doxycycline Erythromycin Gentamicin Erythromycin

Hemiparesis Meningitis Death

Recovery

Penicillin chlortetracycline Penicillin Chloramphenicol None Blood Farmer Gallstones 68 [17]

Medical problems Age (year) Ref.

literature include thrombophlebitis [15], endocarditis [16], with most cases occurring in patients with underlying heart disease, mycotic aneurysm and pericarditis [17±24]. The diagnosis of pericarditis in our patient is certain because of the echocardiographic and the pathologic ®ndings as well as the growth of the organism from the pericardial ¯uid. Pericarditis due to Vibrio fetus has been described in only eight cases since 1966 [17±24] (Table 1). Only two of the eight reported cases had had occupational exposure to cattle. Three had underlying medical problems with lymphoma [19], hypothyroidism [20], and polycystic kidney disease [21]. None of the patients previously described had b-thalassemia. The presentations in most patients were non-speci®c, but often revealed a pleural rub. Most patients responded to a prolonged course of antibiotic (5 weeks). The de®nitive diagnosis in these patients can be challenging to the unaware physician, especially in countries where campylobacter infection has a low incidence. There are no clinical trials comparing the different antibiotic regimens against C. fetus. However, in vitro, the organism is susceptible to gentamicin, which is considered the treatment of choice. For cases with central nervous system involvement, it is recommended to add chloramphenicol for its excellent penetration. The organism is also susceptible to tetracyclines and erythromycin. However, erythromycin resistance has been reported in some isolates, including those reported from Lebanon [13]. Moreover, there are documented cases of treatment failure with erythromycin. Quinolones have good in vitro activity and could be used. The use of penicillins and cephalosporins is not recommended, because of the variable susceptibility results with documented treatment failures, thought to be due to b-lactamase production. However, a more recent report [25] on the susceptibility of 59 isolates of C. fetus subsp. fetus showed that all of them were susceptible to ampicillin, gentamicin, imipenem and meropenem, and none were b-lactamase producers. Twenty-seven per cent of the isolates were resistant to tetracycline. A poor correlation between the in vivo and in vitro results has been noted. The duration of treatment for pericarditis with C. fetus is not well de®ned, but a minimum of 3±4 weeks of antibiotics has been recommended, because of the potential for relapse [23]. With prolonged antimicrobial therapy, the outcome has generally been good with full recovery, as in our patient. In conclusion, though extremely rare, C. fetus pericarditis adds to the list of infectious etiologies occurring in patients with thalassemia. It should be considered in the differential diagnosis of such complications to avoid the delay of accurate identi®cation and appropriate treatment.

1990

1990

1985

1981 1985

1979

1966

1960

R EFER E NCE S Year

Table 1 Cases of Campylobacter fetus pericarditis reported in the literature

Animal exposure

Positive culture

Treatment

Outcome

512 Clinical Microbiology and Infection, Volume 7 Number 9, September 2001

1. Prati D, Zanella A, Farma E et al. A multicenter prospective study on the risk of acquiring liver disease in anti-hepatitis C virus

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3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

negative patients affected from homozygous beta-thalassemia. Blood 1998; 92: 3460±4. Adamkiewicz TV, Berkovitch M, Krishnan C, Polsinelli C, Kermack D, Olivieri NF. Infection due to Yersinia enterolitica in a series of patients with beta-thalassemia: Incidence and predisposing factors. Clin Infect Dis 1998; 27: 1362±6. Jackson N, Zaki M, Rahman AR, Nazim M, Win MN, Osman S. Fatal Campylobacter jejuni infection in a patient splenectomized for thalassemia. J Clin Pathol 1997; 50: 436±7. McFaydean F, Stockman S. Report of the Departmental Committee appointed by the Board of Agriculture and Fisheries to inquire into epizootic abortion, Vol. 3. London: HMSO, 1909. Curtis AH. A motile curved anaerobic bacillus in uterine discharges. J Infect Dis 1913; 12: 165±9. Bokkenheuser V. Vibrio fetus in man. Ten new cases and some epidemiologic observations. Am J Epidemiol 1970; 91: 400±9. Schmidt U, Chmel H, Kaminski Z, Sen P. The clinical spectrum of Campylobacter fetus infections: report of five cases and review of the literature. Q J Med 1980; 49: 431±42. Wyatt RA, Younoszai K, Anuras S, Myers MG. Campylobacter fetus septicemia and hepatitis in a child with agammaglobulinemia. J Pediatr 1977; 91: 441±2. Veron M, Chatelain R. Taxonomic study of the genus campylobacter Sebald and Veron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) SeÂbald and Veron. Int J Syst Bacteriol 1973; 23: 122±34. Rettig PJ. Campylobacter infections in human beings. J Pediatr 1979; 94: 855±64. Taylor PR, Weinstein WM, Bryner JH. Campylobacter fetus infection in human subjects: association with raw milk. Am J Med 1979; 66: 779±83. Guerrant RL, Lahita RG, Winn WC, Roberts RB. Campylobacteriosis in man: pathogenic mechanisms and review of 91 blood stream infections. Am J Med 1978; 65: 584±92.

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13. Talhouk RS, Dana RA, Araj GF, Barbour E, Hashwa F. Prevalence, antimicrobial susceptibility and molecular characterization of Campylobacter isolates recovered from humans and poultry in Lebanon. Leb Med J 1998; 46: 310±16. 14. Tummuru MK, Blaser MJ. Rearrangement of sapA homologs with conserved and variable regions in Campylobacter fetus. Proc Natl Acad Sci USA 1993; 90: 7265±9. 15. Carbone KM, Heinrich MC, Quinn TC. Thrombophlebitis and cellulitis due to Campylobacter fetus ssp. fetus: report of four cases and review of the literature. Medicine 1985; 64: 244±50. 16. Wozniak R, Middleton J, Chmel H, De La Cruz C, Young R. Gram negative endocarditis caused by Campylobacter fetus. South Med J 1978; 71: 1311±12. 17. Jackson JF, Hinton P, Allison F, Jr. Human vibriosis. Report of a patient with relapsing febrile illness due to Vibrio fetus. Am J Med 1960; 28: 986±94. 18. Killam HA, Crowder JG, White AC, Edmonds JH. Pericarditis due to Vibrio fetus. Am J Cardiol 1966; 17: 723±8. 19. Rahman M. Bacteremia and pericarditis from campylobacter infection. Br J Clin Pract 1979; 33: 331±4. 20. Lieber IH, Rensimer ER, Ericsson CD. Campylobacter pericarditis in hypothyroidism. Am Heart J 1981; 102: 462±3. 21. Verresen L, Vrolix M, Verhaegen J, Lins R. Campylobacter bacteremia: report of 2 cases and review of the literature. Acta Clin Belg 1985; 40: 99±104. 22. Gully C, Rioult B, Chambreuil G. Purulent pericarditis caused by Campylobacter fetus ssp fetus. Presse Med 1985; 14: 548. 23. Morrisson VA, Lloyd BK, Chia JK, Tuazon CU. Cardiovascular and bacteremic manifestations of Campylobacter fetus infection: case report and review. Rev Infect Dis 1990; 12: 387±92. 24. Rojo P, Cid M, Latorre M, Alvarez M, Cisterna R. Pericarditis caused by Campylobacter fetus fetus. Enferm Infecc Microbiol Clin 1990; 8: 593±5. 25. Treamblay C, Gaudreau C. Antimicrobial susceptibility testing of 59 strains of Campylobacter fetus subsp fetus. Antimicrob Agents Chemother 1998; 42: 1847±9.

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